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Human herpesviruses types 6 and 7 and febrile seizures
Human Herpesviruses Types 6 and 7 and Febrile Seizures Stephen J. Teach, MD, MPH*, Howard L. Wallace, MS†, Mary Jo Evans, PhD‡, Patricia K. Duffner, MD§, John Hay, PhD†, and Howard S. Faden, MD‡ The frequency was studied with which human herpesviruses types 6 and 7 (HHV-6 and HHV-7) occur in the cerebrospinal fluid (CSF) of patients with febrile seizures and matched control patients. CSF samples were prospectively collected from a case series of patients with febrile seizures and from age-, sex-, and racematched control patients without febrile seizures, all of whom were evaluated in the emergency department of an urban, tertiary care, pediatric medical center. Using polymerase chain reaction, the samples were examined for the presence of viral DNA from HHV-6, HHV-7, herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2), and cytomegalovirus (CMV). CSF from a subset of both groups was also examined for RNA from enteroviruses. During the 7-month, 2-week collection period, a total of 174 patients were evaluated for fever and seizures. Of these, 23 (13.2%) met the study criteria. Their mean age was 1.4 ⴞ 0.7 years. Sixteen (70%) of the 23 were male. The 23 patients were matched to 21 control subjects. None of the samples from the patients or control subjects had polymerase chain reaction evidence of HHV-6, HHV-7, HSV-1, or HSV-2. All samples from the patients were negative for CMV. One control subject was positive for CMV. The 10 patients and seven control subjects tested for enteroviral RNA were negative. Neither HHV-6 nor HHV-7 appears to be present in the CSF of patients with febrile seizures. What role, if any, they have in the pathogenesis of febrile seizures merits further study. © 1999 by Elsevier Science Inc. All rights reserved.
Teach SJ, Wallace HL, Evans MJ, Duffner PK, Hay J, Faden HS. Human herpesviruses types 6 and 7 and febrile seizures. Pediatr Neurol 1999;21:699-703.
From the *Department of Pediatrics; Children’s National Medical Center; George Washington University School of Medicine and Health Sciences; Washington, DC; †Department of Microbiology; State University of New York at Buffalo; ‡Department of Pediatrics; Children’s Hospital of Buffalo; State University of New York at Buffalo School of Medicine and Biomedical Sciences; §Departments of Neurology and Pediatrics; Children’s Hospital of Buffalo; State University of New York at Buffalo School of Medicine and Biomedical Sciences; Buffalo, New York.
Communications should be addressed to: Dr. Teach; Department of Pediatrics; Children’s National Medical Center; 111 Michigan Avenue, NW; Washington, DC 20010. Received March 23, 1999; accepted June 2, 1999.
© 1999 by Elsevier Science Inc. All rights reserved. PII S0887-8994(99)00068-5 ● 0887-8994/99/$20.00
Introduction Recent attention has focused on what role, if any, human herpesviruses types 6 and 7 (HHV-6 and HHV-7) have in the pathogenesis of febrile seizures in young children [1-12]. Data have been conflicting. Certain investigators have speculated that primary HHV-6 and HHV-7 infections occur more frequently in children with febrile seizures than in children with fever and no seizures [5,11]. A recent case-control study, on the other hand, examined HHV-6-specific serologies and used the polymerase chain reaction (PCR) for HHV-6 DNA detection in peripheral blood mononuclear cells and saliva of children with their first or second episode of febrile seizures and age-matched febrile control subjects. There was no difference in the incidence of acute HHV-6 infection in the two groups [12]. If either HHV-6 or HHV-7 has a specific role in inducing febrile seizures, then one might expect to find evidence of them in the cerebrospinal fluid (CSF) of affected patients. Attempts to detect HHV-6 in the CSF, however, have also yielded mixed results. Some authors have reported success using the PCR [2,4,5]; others were unable to recover the intact virus from CSF with culture methods [7,8,12] or to detect DNA with the PCR [8,12]. No attempts have been made to identify HHV-7 in the CSF of children with febrile seizures. To further examine the role of HHV-6, HHV-7, and other neurotropic viruses in the pathogenesis of febrile seizures, the current study used PCR to search for DNA from multiple herpesviruses,
Teach et al: HHV-6 and HHV-7 and Febrile Seizures 699
including HHV-6 and HHV-7, and for RNA from enteroviruses in the CSF of an unselected case series of children with febrile seizures and age-, sex- and race-matched control subjects.
Methods Specimen Collection and Patient Identification. CSF specimens were collected and analyzed at the Children’s Hospital of Buffalo for the 7-month, 2-week period from May 15, 1996 through December 31, 1996. During this period, technicians from the PCR Laboratory collected the excess CSF obtained from all patients in the hospital on a daily basis from the refrigerated (4°C) storage units in the Microbiology, Chemistry, and Hematology Laboratories of the authors’ hospital. Aliquots taken from a single patient at the same time were pooled, and all samples were coded and frozen at ⫺70°C in the PCR Laboratory. Simultaneously, a consecutive case series of patients with febrile seizures, both simple and complex, was assembled from patients treated in the emergency department (ED) of the same institution. Simple febrile seizures were distinguished from complex by duration (less than 15 minutes), lack of focality, and nonrecurrence within 24 hours. Inclusion criteria for patients included a seizure attributed solely to the presence of fever, age between 6 months and 6 years, a temperature of 38°C or greater, collection of CSF at the time of the initial ED encounter, and availability of sufficient stored excess CSF for performance of the PCR. Performance of a lumbar puncture at the time of the initial ED encounter was left solely to the discretion of the attending physician in the ED. Exclusion criteria included a CSF pleocytosis (greater than 7 leukocytes/ mm3 after adjustment for the presence of erythrocytes), an underlying documented seizure disorder or any other chronic underlying neurologic condition, and an insufficient amount of CSF for the PCR. An attempt was then made to match each patient with a control subject of the same age, race, and sex. Control patients underwent CSF sampling for any of a variety of reasons unrelated to febrile seizures. DNA Extraction. An aliquot of 400 L of CSF from each patient was mixed with 400 L of a detergent solution (10 mM Tris, pH 8.3, 2.5 mM MgCl2, 1% Tween 20, and 2% NP-40). Proteinase K was added to a final concentration of 20 g/mL, and the specimen was incubated at 60°C for 1 hour or at 37°C overnight. DNA was then extracted with 800 L of phenol, the aqueous phase recovered and extracted again with 800 L of phenol/chloroform, and the final extract precipitated with 100% ethanol at ⫺70°C for 10 minutes. DNA was collected by centrifugation and dissolved in 400 L of sterile distilled water for amplification. RNA Extraction. An aliquot of 400 L of CSF was extracted with RNA Stat-50 (Teltest, Friendswood, TX) using tRNA at 50 g/mL and glycogen at 100 g/mL as the carrier. The final RNA extract was precipitated with an equal volume of 100% isopropanol, and the precipitate was collected by centrifugation and dissolved in 400 L of sterile distilled water for amplification. Polymerase Chain Reaction. Amplification of DNA was performed at two different laboratories. PCR detection of CMV, HSV-1, and HSV-2 was performed as described previously [13,14]. In brief, in a total volume of 50 L, 30 L of extracted DNA or control solutions in water were amplified in 10 mM Tris, pH 8.3, 1.5 mM MgCl2, 50 mM KCL, 0.01% gelatin, 0.2 mM dNTPs, 0.2 M of both sense and antisense primer sequences, and 1 U of Taq DNA polymerase or AmpliTaq Gold (Perkin Elmer Applied Biosystems, Forest City, CA). An additional control for integrity of the DNA extracted from specimens and for inhibition of the PCR by the specimen was included. This control consisted of amplification of the extracted DNA with primers for the T-cell receptor beta-constant region gene (TcR/c) [13,14]. In addition, each assay included two negative controls: one with uninfected DNA, and one with no DNA. Three concentrations of the appropriate viral DNA at values near the limit of the PCR detection served as positive control samples. Amplification of CMV and of the TcR/c sequences was accomplished with native Taq polymerase, and amplification of the HSV-1 and HSV-2
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sequences was accomplished with AmpliTaq Gold polymerase, which reduced the nonspecific amplification detected. The Perkin Elmer 9600 Thermal Cycler was used, with an initial 5-15 minute denaturation, followed by 35 cycles of 30 seconds at 94°C, 30 seconds at 55°C, and 60 seconds at 72°C. Limits of detection as determined by dilution or by blind proficiency testing were less than 35 viral particles per microliter (HSV-1), less than 66 viral particles per microliter (HSV-2), and less than 29 viral particles per microliter (CMV). Amplification of HHV-6 and HHV-7 was performed under similar conditions [15,16] with the following differences. First, 50 L of DNA extract was assayed in a total volume of 100 L, containing 0.25 M of each primer. Taq Start Antibody (Clontech Laboratories, Palo Alto, CA) was used in conjunction with AmpliTaq DNA polymerase to control nonspecific amplification. Second, cycling conditions were 94°C for 5 minutes, followed by 40 cycles of 94°C for 1 minute, 55°C for 1 minute, and 72°C for 75 seconds. Finally, cycling conditions for HHV-7 also included a 58°C annealing temperature during amplification, and the reaction contained 4.5 mM MgCl2 instead of 1.5 mM as in all the other reactions. Limits of detection for HHV-6 and HHV-7 under this protocol were 25 fg of viral DNA or approximately 100 viral genomes. Reverse Transcriptase PCR for Enteroviruses. Amplification of RNA for the enteroviruses was performed using the rTth DNA polymerase kit (Perkin Elmer Applied Biosytems). This procedure includes reverse transcription at 65°C for 30 minutes, followed by the addition of magnesium and cycling as described above for DNA amplification. Acrylamide Gel Electrophoresis and Scanning. Amplified DNA (20 L) was mixed with 5 L of loading buffer containing 10 g/mL Bromphenol Blue, 50% glycerol, and a 1:20,000 dilution of SYBR Green 1 (Molecular Probes, Eugene, OR). Electrophoresis was performed on 5% acrylamide gels in 0.5 ⫻ Tris:Borate:EDTA buffer for 1 hour at 200 V in 0.5 ⫻ Tris:Borate:EDTA buffer. The gel sandwich was then scanned by a FluorImager (Molecular Dynamics, Sunnyvale, CA), with relative fluorescence calculated by background subtraction.
Results During the 7-month, 2-week collection period, a total of 174 patients were evaluated in the ED for fever and seizures. Forty-six of these patients (26.4%) underwent lumbar puncture during their evaluation, and of these, a total of 23 were eligible for the study. All the patients who underwent lumbar puncture but who were subsequently ineligible were excluded for the same reason—an inadequate amount of CSF for analysis. The 23 eligible patients were matched to 21 control subjects (two patients could not be matched to control subjects). The clinical characteristics of the 23 patients are presented in Table 1. No DNA from HHV-6, HHV-7, HSV-1, HSV-2, or CMV was detected from the CSF of any of the 23 patients (95% confidence interval for detection of each virus from the 23 samples ⫽ 0-13%). No RNA from the enteroviruses was detected in the samples from the 10 patients studied. No DNA from HHV-6, HHV-7, HSV-1, or HSV-2 was detected from the CSF of any of the 21 control subjects. No RNA from the enteroviruses was detected in the samples from the seven control subjects studied. A single control sample collected from a febrile 15month-old male was positive for CMV. This child was evaluated in the ED for fever, upper respiratory tract infection, decreased oral intake, and decreased activity. Physical examination revealed a quiet and noninteractive child with a rectal temperature of 41°C and pharyngeal
Table 1.
Clinical characteristics of 23 study patients
Age (yr) Mean ⫾ S.D. Range (median) Sex Male Female Race White Black Seizure type Complex Simple Prior febrile seizures No Yes Diagnosis in the emergency department Viral syndrome Ottis media Pneumonia Disoposition Discharged Admitted Temperature (°C) Mean ⫾ S.D. Range (median)
1.4 ⫾ 0.7 0.5-3.0 (1.2) 16 7 14 9 15 8 19 4 11 11 1 14 9 39.7 ⫾ 0.6 38.5-40.7 (39.9)
erythema. The peripheral blood leukocyte count was 13,700 cells/mm3, with 41% polymorphonuclear forms and 22% band forms. Lumbar puncture revealed no CSF pleocytosis. Bacterial cultures of blood, CSF, and urine were negative. A viral culture of the CSF was negative. The patient’s past medical history, growth, and development were reviewed with his personal physician. No abnormalities were identified. Discussion HHV-6 and HHV-7 are closely related, lymphotropic, double-strand DNA viruses that share sequence homology and antigenic properties [17,18]. Each is a cause of uncomplicated febrile illness in young children [5,11], and each causes exanthem subitum [19,20]. Both also appear to have tropism for the central nervous system, and primary infections with both have been linked to neurologic sequelae in children: HHV-6 to meningoencephalitis [21-26] and transverse myelitis [27] and HHV-7 to acute hemiplegia [8]. HHV-6 has been demonstrated to infect human astrocytes [28] and to persist in the CSF after primary infection [4]. Certain investigators have suggested that both HHV-6 [2,5] and HHV-7 [11] are causally linked to uncomplicated febrile seizures, although the exact mechanism remains obscure. Between 3% and 18% of patients with primary HHV-6 infection [1,3-5,11] and 6-75% of patients with primary HHV-7 infection [6,9,11] have febrile seizures during their acute illness. At least one study demonstrated that a significantly greater proportion of febrile children between 12 and 15 months of age with primary HHV-6 infection had febrile seizures than did febrile
control subjects without HHV-6 infection (8 [36%] of 22 vs 17 [13%] of 131, respectively, P ⫽ 0.01) [5]. The inverse, and ultimately more important question, however, is whether primary infections with HHV-6 and HHV-7 occur more commonly in febrile patients with seizures than in febrile patients without seizures. No such studies have been done for HHV-7 infections, but previous studies have demonstrated that 26-43% of febrile seizures are associated with primary HHV-6 infection [5,7,12]. A recent matched case-control study comparing febrile children 6 months to 2 years of age with and without seizures, however, presented no evidence of a difference between the two groups in the incidence of primary HHV-6 infection (15 [43%] of 35 vs 15 (45%) of 33, respectively, odds ratio ⫽ 0.9, 95% confidence interval 0.3-2.3) [12]. If febrile seizures are caused, at least in part, by some direct effect of infections with either HHV-6 or HHV-7, then one might expect to detect the viral genome or intact virus in the CSF of affected patients. Using the previously described incidence of primary HHV-6 infection among children with febrile seizures (26-43%) [5,7,12], we would have expected to find HHV-6 genome in six to 10 of the authors’ 23 patients with febrile seizures. Instead, the authors were unable to detect the genome in any of these samples or from the CSF samples from the age-, sex-, and race-matched control subjects. Several explanations for these results are possible. First, these results may have occurred solely by chance. The upper limit of the 95% confidence interval for positive detection of DNA from either HHV-6 or HHV-7 from the authors’ 23 samples was 13%, or three samples. Chance seems unlikely, however, given the authors’ expectation of detecting HHV-6 in six to 10 samples. Second, the age distribution of the authors’ patients may have resulted in a selection bias against HHV-6 detection. The median age of primary HHV-6 infection is 8-9 months [5,11]. Only nine (39%) of the authors’ patients were younger than 12 months old. The median age of primary HHV-7 infection, however, is later (range ⫽ 16-26 months) [6,11]. Third, the authors’ PCR may have been insufficiently sensitive to detect low levels of viral genome. Both HHV-6 and HHV-7 are primarily lymphocyte associated, and no patient in the authors’ series had CSF pleocytosis (Table 2). Fourth, the viral genome may pass through the CSF either before or after the seizure occurs. Fifth, the seizure may occur without direct viral invasion of the central nervous system or the CSF but instead may occur because of a viral-induced vasculitis or a toxin liberated as part of the viral infection. Finally, febrile seizures may occur independently of HHV-6 and HHV-7 infection. In other words, the seizures were induced by the fever caused by the infections and not by some specific neurotropic property unique to the viruses. Arguing against the latter two explanations is that some groups have reported successful detection of HHV-6 from CSF of a small number of patients with febrile seizures using the PCR. Caserta et al. [4], for example, detected
Teach et al: HHV-6 and HHV-7 and Febrile Seizures 701
Table 2.
Laboratory values for 23 study patients
Peripheral blood leukocyte count (⫻103/mm3) CSF leukocyte count (⫻103/mm3) CSF erythrocyte count (⫻103/mm3) CSF protein (mg/dL) CSF glucose (mg/dL) Urine culture Blood culture CSF bacterial culture CSF viral culture Chest x-ray
5.7 ⫾ 8.4 (5.7-30.6, 13.6) 1.9 ⫾ 2.7 (0-13, 1) 320.7 ⫾ 1383 (0-6,500, 1) 17.7 ⫾ 8.6 (7-37, 14) 78.4 ⫾ 21.9 (35-122, 77) 1/18*† ‡ 1/23 0/23 0/12 1/8
Data presented as mean ⫾ S.D., with the range and median in parentheses, unless otherwise noted. * Number positive of total sampled. † Greater than 105 colonies of an enteric Gram-negative rod. ‡ Streptococcus pneumoniae.
DNA from HHV-6 from the CSF of two such patients younger than 3 years of age with primary HHV-6 infection and 11 such patients with evidence of previous HHV-6 infection. Kondo et al. [2] detected HHV-6 DNA from one of seven CSF samples from children with a first febrile seizure, and from eight of 10 samples from eight children with recurrent febrile seizures. Hall et al. [5] reported detection of DNA from HHV-6 in two of seven patients with febrile seizures (primary or recurrent not specified). The role of HHV-6 or HHV-7 in the pathogenesis of recurrent febrile seizures is even more unclear, with some authors speculating that they have an increased role [2] and others speculating that they have a decreased role [11]. The authors studied 19 children with their first febrile seizure and four children with a recurrent febrile seizure and were unable to detect HHV-6 or HHV-7 in any of the CSF samples. Other groups have reported results of HHV-6 detection similar to the authors’ results. Hukin et al. [12], for example, was unable to detect HHV-6 in the CSF of 15 patients with febrile seizures either by PCR or viral culture. Other groups have reported similar results [7,8]. No study before the authors’ study attempted detection of HHV-7. Why some groups have successfully detected HHV-6 and others have not is unclear. Possible explanations include the sensitivity and specificity of the particular assays, the number of primary vs recurrent seizures studied, the ages of the patients studied, or the effects of random sampling. This study has two important limitations. First, we did not attempt to establish whether these patients had primary infection with HHV-6 or HHV-7 by performing either serologic testing or PCR of peripheral blood mononuclear cells or of plasma. As previously indicated, it is therefore possible that none of the authors’ patients had primary HHV-6 or HHV-7 infection. Second, we deliberately excluded patients with CSF pleocytosis and therefore cannot comment on the presence or absence of HHV-6 and HHV-7 infection in children with meningoencephalitis.
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Excluding patients with CSF pleocytosis was necessary, however, if patients in the present case series were to meet the definition of having a febrile seizure. In conclusion, this study was unable to document viral genome from HHV-6, HHV-7, CMV, HSV-1, or HSV-2 in the CSF of 23 patients with febrile seizures. The role, if any, of HHV-6 and HHV-7 in the pathogenesis of febrile seizures merits further study.
References [1] Pruksananonda P, Hall CB, Insel RA, et al. Primary human herpesvirus-6 infection in young children. N Engl J Med 1992;326:1445-50. [2] Kondo K, Nagafuji H, Hata A, Tomomori C, Yamanishi K. Association of human herpesvirus 6 infection of the central nervous system with recurrence of febrile convuslions. J Infect Dis 1993;167:1197-200. [3] Asano Y, Yoshikawa T, Suga S, et al. Clinical features of infants with primary human herpesvirus-6 infection. Pediatrics 1994;93:104-8. [4] Caserta MT, Hall CB, Schnabel K, et al. Neuroinvasion and persistence of human herpesvirus-6 in children. J Infect Dis 1994;170: 1586-9. [5] Hall CB, Long CE, Schnabel KC, et al. Human herpesvirus-6 infection in children. N Engl J Med 1994;331:432-8. [6] Torigoe S, Kumamoto T, Koide W, Taya K, Yamanishi K. Clinical manifestations associated with human herpesvirus 7 infection. Arch Dis Child 1995;72:518-9. [7] Barone S, Kaplan MH, Krilov LR. Human herpesvirus-6 infection in children with first febrile seizures. J Pediatr 1995;127:95-7. [8] Torigoe S, Koide W, Yamada M, Miyashiro E, Tanaka-Taya K, Yamanishi K. Human herpesvirus 7 infection associated with central nervous system manifestations. J Pediatr 1996;129:301-5. [9] Suga S, Yoshikawa T, Nagai T, Asano Y. Clinical features and virological findings in children with primary human herpesvirus 7 infection. Pediatrics 1997;99:e4. [10] 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. [11] Caserta MT, Hall CB, Schnabel K, Long CE, D’Heron N. Primary human herpesvirus 7 infection: A comparison of human herpesvirus 7 and human herpesvirus 6 infections in children. J Pediatr 1998;133:386-9. [12] Hukin J, Farrell K, MacWilliams LM, et al. Case control study of primary human herpesvirus 6 infection in children with febrile seizures. Pediatrics 1998;101:e3. [13] Evans M, Edward-Spring Y, Povinelli D, et al. Rapid, nonradioactive detection of virus infection by polymerase chain reaction. Clin Diagn Virol 1996;6:163-74. [14] Evans M, Edwards-Spring Y, Meyers J, et al. Polymerase chain reaction for the detection of cytomegalovirus in organ and bone marrow transplant recipients. Immunol Invest 1997;26:209-29. [15] Jarrett RF, Clark DA, Josephs SF, Onions DE. Detection of human herpesvirus-6 DNA in peripheral blood and saliva. J Med Virol 1996;32:73-6. [16] Wallace HL, Natelson B, Gause W, Hay J. An evaluation of human herpesviruses in chronic fatigue syndrome. Clin Diagn Lab Immunol 1999;6:216-23. [17] Benerman ZN, Ablashi DV, Li G, et al. Human herpesvirus-7 is a T-lymphotropic and is related to, but significantly different from human herpesvirus-6 and human cytomegalovirus. Proc Natl Acad Sci U S A 1992;89:10552-6. [18] Benerman ZN, Gallo RC, Ablashi DV, Frenkel N, Katsafanas G, Kramarsky B. Human herpesvirus-7 (HHV-7) strain JI: Independent confirmation of HHV-7. J Infect Dis 1992:166:690-1. [19] Yamanishi K, Okuno T, Shiraki K. Identification of human herpesvirus 6 as a causal agent fro exanthem subitum. Lancet 1988;1: 1065-7.
[20] Tanaka K, Kondo T, Torigoe S, Okada S, Mukai T, Yamanishi K. Human herpesvirus 7: Another causal agent for roseola (exanthem subitum). J Pediatr 1994;125:1-5. [21] Ishiguro N, Yamada S, Takahashi T, et al. Meningo-encephalitis associated with HHV-6 related exanthem subitum. Acta Paediatr Scand 1990;79:987-9. [22] Huang L, Lee C, Lee P, Chen J, Wang P. Meningitis caused by human herpesvirus-6. Arch Dis Child 1991;66:1444-5. [23] Asano Y, Yoshikawa T, Kajita Y, et al. Fatal encephalitis/ encephalopathy in primary human herpesvirus-6 infection. Arch Dis Child 1992;67:1484-5. [24] Yanagihara K, Tanaka-Taya K, Itagaki Y, et al. Human
herpesvirus-6 meningoencephalitis with sequelae. Pediatr Infect Dis J 1995;14:240-1. [25] Yoshikawa T, Nakashima T, Suga S, et al. Human herpesvirus-6 DNA in cerebrospinal fluid of a child with exanthem subitum and meningoencephalitis. Pediatrics 1992;89:888-90. [26] McCullers JA, Lakeman FD, Whitley R. Human herpesvirus 6 is associated with focal encephalitis. Clin Infect Dis 1995;21:571-6. [27] Hill AE, Hicks EM, Coyle PV. Human herpesvirus 6 and central nervous system complications. Dev Med Child Neurol 1994;36:651-2. [28] He J, McCarthy M, Zhou Y, Chandran B, Wood C. Infection of primary human fetal astrocytes by human herpesvirus-6. J Virol 1996; 70:1296-300.
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Teach SJ, Wallace HL, Evans MJ, Duffner PK, Hay J, Faden HS. Human herpesviruses types 6 and 7 and febrile seizures. Pediatr Neurol 1999;21:699-703.
From the *Department of Pediatrics; Children’s National Medical Center; George Washington University School of Medicine and Health Sciences; Washington, DC; †Department of Microbiology; State University of New York at Buffalo; ‡Department of Pediatrics; Children’s Hospital of Buffalo; State University of New York at Buffalo School of Medicine and Biomedical Sciences; §Departments of Neurology and Pediatrics; Children’s Hospital of Buffalo; State University of New York at Buffalo School of Medicine and Biomedical Sciences; Buffalo, New York.
Communications should be addressed to: Dr. Teach; Department of Pediatrics; Children’s National Medical Center; 111 Michigan Avenue, NW; Washington, DC 20010. Received March 23, 1999; accepted June 2, 1999.
© 1999 by Elsevier Science Inc. All rights reserved. PII S0887-8994(99)00068-5 ● 0887-8994/99/$20.00
Introduction Recent attention has focused on what role, if any, human herpesviruses types 6 and 7 (HHV-6 and HHV-7) have in the pathogenesis of febrile seizures in young children [1-12]. Data have been conflicting. Certain investigators have speculated that primary HHV-6 and HHV-7 infections occur more frequently in children with febrile seizures than in children with fever and no seizures [5,11]. A recent case-control study, on the other hand, examined HHV-6-specific serologies and used the polymerase chain reaction (PCR) for HHV-6 DNA detection in peripheral blood mononuclear cells and saliva of children with their first or second episode of febrile seizures and age-matched febrile control subjects. There was no difference in the incidence of acute HHV-6 infection in the two groups [12]. If either HHV-6 or HHV-7 has a specific role in inducing febrile seizures, then one might expect to find evidence of them in the cerebrospinal fluid (CSF) of affected patients. Attempts to detect HHV-6 in the CSF, however, have also yielded mixed results. Some authors have reported success using the PCR [2,4,5]; others were unable to recover the intact virus from CSF with culture methods [7,8,12] or to detect DNA with the PCR [8,12]. No attempts have been made to identify HHV-7 in the CSF of children with febrile seizures. To further examine the role of HHV-6, HHV-7, and other neurotropic viruses in the pathogenesis of febrile seizures, the current study used PCR to search for DNA from multiple herpesviruses,
Teach et al: HHV-6 and HHV-7 and Febrile Seizures 699
including HHV-6 and HHV-7, and for RNA from enteroviruses in the CSF of an unselected case series of children with febrile seizures and age-, sex- and race-matched control subjects.
Methods Specimen Collection and Patient Identification. CSF specimens were collected and analyzed at the Children’s Hospital of Buffalo for the 7-month, 2-week period from May 15, 1996 through December 31, 1996. During this period, technicians from the PCR Laboratory collected the excess CSF obtained from all patients in the hospital on a daily basis from the refrigerated (4°C) storage units in the Microbiology, Chemistry, and Hematology Laboratories of the authors’ hospital. Aliquots taken from a single patient at the same time were pooled, and all samples were coded and frozen at ⫺70°C in the PCR Laboratory. Simultaneously, a consecutive case series of patients with febrile seizures, both simple and complex, was assembled from patients treated in the emergency department (ED) of the same institution. Simple febrile seizures were distinguished from complex by duration (less than 15 minutes), lack of focality, and nonrecurrence within 24 hours. Inclusion criteria for patients included a seizure attributed solely to the presence of fever, age between 6 months and 6 years, a temperature of 38°C or greater, collection of CSF at the time of the initial ED encounter, and availability of sufficient stored excess CSF for performance of the PCR. Performance of a lumbar puncture at the time of the initial ED encounter was left solely to the discretion of the attending physician in the ED. Exclusion criteria included a CSF pleocytosis (greater than 7 leukocytes/ mm3 after adjustment for the presence of erythrocytes), an underlying documented seizure disorder or any other chronic underlying neurologic condition, and an insufficient amount of CSF for the PCR. An attempt was then made to match each patient with a control subject of the same age, race, and sex. Control patients underwent CSF sampling for any of a variety of reasons unrelated to febrile seizures. DNA Extraction. An aliquot of 400 L of CSF from each patient was mixed with 400 L of a detergent solution (10 mM Tris, pH 8.3, 2.5 mM MgCl2, 1% Tween 20, and 2% NP-40). Proteinase K was added to a final concentration of 20 g/mL, and the specimen was incubated at 60°C for 1 hour or at 37°C overnight. DNA was then extracted with 800 L of phenol, the aqueous phase recovered and extracted again with 800 L of phenol/chloroform, and the final extract precipitated with 100% ethanol at ⫺70°C for 10 minutes. DNA was collected by centrifugation and dissolved in 400 L of sterile distilled water for amplification. RNA Extraction. An aliquot of 400 L of CSF was extracted with RNA Stat-50 (Teltest, Friendswood, TX) using tRNA at 50 g/mL and glycogen at 100 g/mL as the carrier. The final RNA extract was precipitated with an equal volume of 100% isopropanol, and the precipitate was collected by centrifugation and dissolved in 400 L of sterile distilled water for amplification. Polymerase Chain Reaction. Amplification of DNA was performed at two different laboratories. PCR detection of CMV, HSV-1, and HSV-2 was performed as described previously [13,14]. In brief, in a total volume of 50 L, 30 L of extracted DNA or control solutions in water were amplified in 10 mM Tris, pH 8.3, 1.5 mM MgCl2, 50 mM KCL, 0.01% gelatin, 0.2 mM dNTPs, 0.2 M of both sense and antisense primer sequences, and 1 U of Taq DNA polymerase or AmpliTaq Gold (Perkin Elmer Applied Biosystems, Forest City, CA). An additional control for integrity of the DNA extracted from specimens and for inhibition of the PCR by the specimen was included. This control consisted of amplification of the extracted DNA with primers for the T-cell receptor beta-constant region gene (TcR/c) [13,14]. In addition, each assay included two negative controls: one with uninfected DNA, and one with no DNA. Three concentrations of the appropriate viral DNA at values near the limit of the PCR detection served as positive control samples. Amplification of CMV and of the TcR/c sequences was accomplished with native Taq polymerase, and amplification of the HSV-1 and HSV-2
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sequences was accomplished with AmpliTaq Gold polymerase, which reduced the nonspecific amplification detected. The Perkin Elmer 9600 Thermal Cycler was used, with an initial 5-15 minute denaturation, followed by 35 cycles of 30 seconds at 94°C, 30 seconds at 55°C, and 60 seconds at 72°C. Limits of detection as determined by dilution or by blind proficiency testing were less than 35 viral particles per microliter (HSV-1), less than 66 viral particles per microliter (HSV-2), and less than 29 viral particles per microliter (CMV). Amplification of HHV-6 and HHV-7 was performed under similar conditions [15,16] with the following differences. First, 50 L of DNA extract was assayed in a total volume of 100 L, containing 0.25 M of each primer. Taq Start Antibody (Clontech Laboratories, Palo Alto, CA) was used in conjunction with AmpliTaq DNA polymerase to control nonspecific amplification. Second, cycling conditions were 94°C for 5 minutes, followed by 40 cycles of 94°C for 1 minute, 55°C for 1 minute, and 72°C for 75 seconds. Finally, cycling conditions for HHV-7 also included a 58°C annealing temperature during amplification, and the reaction contained 4.5 mM MgCl2 instead of 1.5 mM as in all the other reactions. Limits of detection for HHV-6 and HHV-7 under this protocol were 25 fg of viral DNA or approximately 100 viral genomes. Reverse Transcriptase PCR for Enteroviruses. Amplification of RNA for the enteroviruses was performed using the rTth DNA polymerase kit (Perkin Elmer Applied Biosytems). This procedure includes reverse transcription at 65°C for 30 minutes, followed by the addition of magnesium and cycling as described above for DNA amplification. Acrylamide Gel Electrophoresis and Scanning. Amplified DNA (20 L) was mixed with 5 L of loading buffer containing 10 g/mL Bromphenol Blue, 50% glycerol, and a 1:20,000 dilution of SYBR Green 1 (Molecular Probes, Eugene, OR). Electrophoresis was performed on 5% acrylamide gels in 0.5 ⫻ Tris:Borate:EDTA buffer for 1 hour at 200 V in 0.5 ⫻ Tris:Borate:EDTA buffer. The gel sandwich was then scanned by a FluorImager (Molecular Dynamics, Sunnyvale, CA), with relative fluorescence calculated by background subtraction.
Results During the 7-month, 2-week collection period, a total of 174 patients were evaluated in the ED for fever and seizures. Forty-six of these patients (26.4%) underwent lumbar puncture during their evaluation, and of these, a total of 23 were eligible for the study. All the patients who underwent lumbar puncture but who were subsequently ineligible were excluded for the same reason—an inadequate amount of CSF for analysis. The 23 eligible patients were matched to 21 control subjects (two patients could not be matched to control subjects). The clinical characteristics of the 23 patients are presented in Table 1. No DNA from HHV-6, HHV-7, HSV-1, HSV-2, or CMV was detected from the CSF of any of the 23 patients (95% confidence interval for detection of each virus from the 23 samples ⫽ 0-13%). No RNA from the enteroviruses was detected in the samples from the 10 patients studied. No DNA from HHV-6, HHV-7, HSV-1, or HSV-2 was detected from the CSF of any of the 21 control subjects. No RNA from the enteroviruses was detected in the samples from the seven control subjects studied. A single control sample collected from a febrile 15month-old male was positive for CMV. This child was evaluated in the ED for fever, upper respiratory tract infection, decreased oral intake, and decreased activity. Physical examination revealed a quiet and noninteractive child with a rectal temperature of 41°C and pharyngeal
Table 1.
Clinical characteristics of 23 study patients
Age (yr) Mean ⫾ S.D. Range (median) Sex Male Female Race White Black Seizure type Complex Simple Prior febrile seizures No Yes Diagnosis in the emergency department Viral syndrome Ottis media Pneumonia Disoposition Discharged Admitted Temperature (°C) Mean ⫾ S.D. Range (median)
1.4 ⫾ 0.7 0.5-3.0 (1.2) 16 7 14 9 15 8 19 4 11 11 1 14 9 39.7 ⫾ 0.6 38.5-40.7 (39.9)
erythema. The peripheral blood leukocyte count was 13,700 cells/mm3, with 41% polymorphonuclear forms and 22% band forms. Lumbar puncture revealed no CSF pleocytosis. Bacterial cultures of blood, CSF, and urine were negative. A viral culture of the CSF was negative. The patient’s past medical history, growth, and development were reviewed with his personal physician. No abnormalities were identified. Discussion HHV-6 and HHV-7 are closely related, lymphotropic, double-strand DNA viruses that share sequence homology and antigenic properties [17,18]. Each is a cause of uncomplicated febrile illness in young children [5,11], and each causes exanthem subitum [19,20]. Both also appear to have tropism for the central nervous system, and primary infections with both have been linked to neurologic sequelae in children: HHV-6 to meningoencephalitis [21-26] and transverse myelitis [27] and HHV-7 to acute hemiplegia [8]. HHV-6 has been demonstrated to infect human astrocytes [28] and to persist in the CSF after primary infection [4]. Certain investigators have suggested that both HHV-6 [2,5] and HHV-7 [11] are causally linked to uncomplicated febrile seizures, although the exact mechanism remains obscure. Between 3% and 18% of patients with primary HHV-6 infection [1,3-5,11] and 6-75% of patients with primary HHV-7 infection [6,9,11] have febrile seizures during their acute illness. At least one study demonstrated that a significantly greater proportion of febrile children between 12 and 15 months of age with primary HHV-6 infection had febrile seizures than did febrile
control subjects without HHV-6 infection (8 [36%] of 22 vs 17 [13%] of 131, respectively, P ⫽ 0.01) [5]. The inverse, and ultimately more important question, however, is whether primary infections with HHV-6 and HHV-7 occur more commonly in febrile patients with seizures than in febrile patients without seizures. No such studies have been done for HHV-7 infections, but previous studies have demonstrated that 26-43% of febrile seizures are associated with primary HHV-6 infection [5,7,12]. A recent matched case-control study comparing febrile children 6 months to 2 years of age with and without seizures, however, presented no evidence of a difference between the two groups in the incidence of primary HHV-6 infection (15 [43%] of 35 vs 15 (45%) of 33, respectively, odds ratio ⫽ 0.9, 95% confidence interval 0.3-2.3) [12]. If febrile seizures are caused, at least in part, by some direct effect of infections with either HHV-6 or HHV-7, then one might expect to detect the viral genome or intact virus in the CSF of affected patients. Using the previously described incidence of primary HHV-6 infection among children with febrile seizures (26-43%) [5,7,12], we would have expected to find HHV-6 genome in six to 10 of the authors’ 23 patients with febrile seizures. Instead, the authors were unable to detect the genome in any of these samples or from the CSF samples from the age-, sex-, and race-matched control subjects. Several explanations for these results are possible. First, these results may have occurred solely by chance. The upper limit of the 95% confidence interval for positive detection of DNA from either HHV-6 or HHV-7 from the authors’ 23 samples was 13%, or three samples. Chance seems unlikely, however, given the authors’ expectation of detecting HHV-6 in six to 10 samples. Second, the age distribution of the authors’ patients may have resulted in a selection bias against HHV-6 detection. The median age of primary HHV-6 infection is 8-9 months [5,11]. Only nine (39%) of the authors’ patients were younger than 12 months old. The median age of primary HHV-7 infection, however, is later (range ⫽ 16-26 months) [6,11]. Third, the authors’ PCR may have been insufficiently sensitive to detect low levels of viral genome. Both HHV-6 and HHV-7 are primarily lymphocyte associated, and no patient in the authors’ series had CSF pleocytosis (Table 2). Fourth, the viral genome may pass through the CSF either before or after the seizure occurs. Fifth, the seizure may occur without direct viral invasion of the central nervous system or the CSF but instead may occur because of a viral-induced vasculitis or a toxin liberated as part of the viral infection. Finally, febrile seizures may occur independently of HHV-6 and HHV-7 infection. In other words, the seizures were induced by the fever caused by the infections and not by some specific neurotropic property unique to the viruses. Arguing against the latter two explanations is that some groups have reported successful detection of HHV-6 from CSF of a small number of patients with febrile seizures using the PCR. Caserta et al. [4], for example, detected
Teach et al: HHV-6 and HHV-7 and Febrile Seizures 701
Table 2.
Laboratory values for 23 study patients
Peripheral blood leukocyte count (⫻103/mm3) CSF leukocyte count (⫻103/mm3) CSF erythrocyte count (⫻103/mm3) CSF protein (mg/dL) CSF glucose (mg/dL) Urine culture Blood culture CSF bacterial culture CSF viral culture Chest x-ray
5.7 ⫾ 8.4 (5.7-30.6, 13.6) 1.9 ⫾ 2.7 (0-13, 1) 320.7 ⫾ 1383 (0-6,500, 1) 17.7 ⫾ 8.6 (7-37, 14) 78.4 ⫾ 21.9 (35-122, 77) 1/18*† ‡ 1/23 0/23 0/12 1/8
Data presented as mean ⫾ S.D., with the range and median in parentheses, unless otherwise noted. * Number positive of total sampled. † Greater than 105 colonies of an enteric Gram-negative rod. ‡ Streptococcus pneumoniae.
DNA from HHV-6 from the CSF of two such patients younger than 3 years of age with primary HHV-6 infection and 11 such patients with evidence of previous HHV-6 infection. Kondo et al. [2] detected HHV-6 DNA from one of seven CSF samples from children with a first febrile seizure, and from eight of 10 samples from eight children with recurrent febrile seizures. Hall et al. [5] reported detection of DNA from HHV-6 in two of seven patients with febrile seizures (primary or recurrent not specified). The role of HHV-6 or HHV-7 in the pathogenesis of recurrent febrile seizures is even more unclear, with some authors speculating that they have an increased role [2] and others speculating that they have a decreased role [11]. The authors studied 19 children with their first febrile seizure and four children with a recurrent febrile seizure and were unable to detect HHV-6 or HHV-7 in any of the CSF samples. Other groups have reported results of HHV-6 detection similar to the authors’ results. Hukin et al. [12], for example, was unable to detect HHV-6 in the CSF of 15 patients with febrile seizures either by PCR or viral culture. Other groups have reported similar results [7,8]. No study before the authors’ study attempted detection of HHV-7. Why some groups have successfully detected HHV-6 and others have not is unclear. Possible explanations include the sensitivity and specificity of the particular assays, the number of primary vs recurrent seizures studied, the ages of the patients studied, or the effects of random sampling. This study has two important limitations. First, we did not attempt to establish whether these patients had primary infection with HHV-6 or HHV-7 by performing either serologic testing or PCR of peripheral blood mononuclear cells or of plasma. As previously indicated, it is therefore possible that none of the authors’ patients had primary HHV-6 or HHV-7 infection. Second, we deliberately excluded patients with CSF pleocytosis and therefore cannot comment on the presence or absence of HHV-6 and HHV-7 infection in children with meningoencephalitis.
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Excluding patients with CSF pleocytosis was necessary, however, if patients in the present case series were to meet the definition of having a febrile seizure. In conclusion, this study was unable to document viral genome from HHV-6, HHV-7, CMV, HSV-1, or HSV-2 in the CSF of 23 patients with febrile seizures. The role, if any, of HHV-6 and HHV-7 in the pathogenesis of febrile seizures merits further study.
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[20] Tanaka K, Kondo T, Torigoe S, Okada S, Mukai T, Yamanishi K. Human herpesvirus 7: Another causal agent for roseola (exanthem subitum). J Pediatr 1994;125:1-5. [21] Ishiguro N, Yamada S, Takahashi T, et al. Meningo-encephalitis associated with HHV-6 related exanthem subitum. Acta Paediatr Scand 1990;79:987-9. [22] Huang L, Lee C, Lee P, Chen J, Wang P. Meningitis caused by human herpesvirus-6. Arch Dis Child 1991;66:1444-5. [23] Asano Y, Yoshikawa T, Kajita Y, et al. Fatal encephalitis/ encephalopathy in primary human herpesvirus-6 infection. Arch Dis Child 1992;67:1484-5. [24] Yanagihara K, Tanaka-Taya K, Itagaki Y, et al. Human
herpesvirus-6 meningoencephalitis with sequelae. Pediatr Infect Dis J 1995;14:240-1. [25] Yoshikawa T, Nakashima T, Suga S, et al. Human herpesvirus-6 DNA in cerebrospinal fluid of a child with exanthem subitum and meningoencephalitis. Pediatrics 1992;89:888-90. [26] McCullers JA, Lakeman FD, Whitley R. Human herpesvirus 6 is associated with focal encephalitis. Clin Infect Dis 1995;21:571-6. [27] Hill AE, Hicks EM, Coyle PV. Human herpesvirus 6 and central nervous system complications. Dev Med Child Neurol 1994;36:651-2. [28] He J, McCarthy M, Zhou Y, Chandran B, Wood C. Infection of primary human fetal astrocytes by human herpesvirus-6. J Virol 1996; 70:1296-300.
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