Journal Pre-proof

Clinical features of complex febrile seizure caused by primary human herpesvirus 6B

infection

Misa Miyake, MD, Yoshiki Kawamura, MD, PhD, Fumihiko Hattori, MD, PhD, Hiroki
Miura, MD, PhD, Naoko Ishihara, MD, PhD, Tetsushi Yoshikawa, MD, PhD

PII: S0887-8994(20)30109-0
DOI: https://doi.org/10.1016/j.pediatrneurol.2020.03.015
Reference: PNU 9755

To appear in: Pediatric Neurology

Received Date: 6 December 2019

Revised Date: 29 January 2020
Accepted Date: 14 March 2020

Please cite this article as: Miyake M, Kawamura Y, Hattori F, Miura H, Ishihara N, Yoshikawa T, Clinical
features of complex febrile seizure caused by primary human herpesvirus 6B infection, Pediatric
Neurology (2020), doi: https://doi.org/10.1016/j.pediatrneurol.2020.03.015.

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© 2020 Elsevier Inc. All rights reserved.

 1 Revised manuscript: PNU-D-19-00227R1

2 Original Article

3 Clinical features of complex febrile seizure caused by primary human herpesvirus 6B

4 infection

5 Misa Miyake, MD a, Yoshiki Kawamura, MD, PhD a, *

; Fumihiko Hattori, MD, PhD a, b
;

6 Hiroki Miura, MD, PhD a; Naoko Ishihara, MD, PhD a; Tetsushi Yoshikawa, MD, PhD a

a
7 Department of Pediatrics, Fujita Health University School of Medicine, Toyoake Japan

b
8 Department of Pediatrics, Kariya Toyota General Hospital, Kariya Japan

10 Declaration of interest: none.

11 Funding: This work was supported by JSPS KAKENHI Grant Number JP 19K17346 and The

12 Mother and Child Health Foundation Grant Number 30-10.

13

14 * Communications should be addressed to: Kawamura; Department of Pediatrics; Fujita

15 Health University School of Medicine; 1-98 Dengakugakubo, Kutsukake-cho, Toyoake,

16 Japan 470-1192.

17 E-mail address: yoshiki@fujita-hu.ac.jp (Y. Kawamura).

18 Word count: 1751 words

19

1
20 Abstract

21 Background: It is well known that febrile seizure is common complication of exanthem

22 subitum. In this study, we compared the clinical features and backgrounds of complex febrile

23 seizure patients with and without primary HHV-6B infection.

24 Methods: Sixty-two patients were enrolled after experiencing their first febrile seizure.

25 Primary HHV-6B infection was confirmed when HHV-6B DNA was detected and HHV-6B

26 antibody was negative in serum obtained during the acute phase of infection. Patient

27 backgrounds such as age, gender and features of seizures were evaluated between patients

28 with and without HHV-6B infection.

29 Results: Thirty complex febrile seizure patients were diagnosed with primary HHV-6B

30 infection. Those with primary HHV-6B infection (median, 13 months; range, 7–39 months)

31 were significantly younger than those without primary HHV-6B infection (median, 19

32 months; range, 10–59 months) (P=0.001), and the proportion of males was significantly

33 higher in patients without primary HHV-6B infection (male/female, 25/7) than in those with

34 the infection (male/female, 14/16) (P=0.017). An interval between fever onset and seizures

35 more than 24 hours after fever onset were significantly more common in the patients with

36 primary HHV-6B infection (15 of the 30 patients) than in those without primary HHV-6B

37 infection (2 of the 32 patients) (P <0.001).

2
38 Conclusions: The younger age at onset, the different gender ratio with febrile seizure due to

39 other causes, and the length of interval between fever and seizures were features of complex

40 febrile seizure associated HHV-6B infection. These findings may suggest a different

41 mechanism of complex febrile seizure onset with due to other causes.

42 Keywords:

43 Human herpesvirus 6B, Febrile seizure, Exanthem subitum

3
44 Introduction

45 Exanthem subitum is a common infectious disease in infants and young children,

46 and is caused by primary human herpesvirus 6B (HHV-6B) infection.1,2 It is well known that

47 febrile seizure (FS) is common complication of exanthem subitum.3 Previous reports

48 suggested that the virus can invade the central nervous system in some patients with

49 exanthem subitum and cause encephalitis or other central nervous system complications.4-7

50 Viral DNA is detected in cerebrospinal fluid (CSF) collected from patients with primary

51 HHV-6B infection and also FS, bulging fontanelle, and encephalitis.8-11 Although high copy

52 numbers of HHV-6B DNA have been detected in CSF collected from patients with

53 post-transplant HHV-6B encephalitis,12-15 our previous study showed that in primary

54 HHV-6B infection, small amounts of viral DNA were present in approximately 32% and 17%

55 of CSF samples collected from patients with HHV-6 encephalitis and FS associated with

56 HHV-6B, respectively.16 Therefore, it is considered that direct viral replication in brain tissue

57 is not the main pathological mechanism for encephalitis or FS at the time of primary HHV-6B

58 infection.

59 It has been demonstrated that primary HHV-6B infection is frequently associated

60 with FS in infants and young children, and that it often results in the development of complex

61 febrile seizure (CFS) and might be a risk factor for the subsequent development of epilepsy.17

62 Additionally, HHV-6 DNA has been detected more frequently in CSF collected from patients

4
63 with repeated episodes of FS than those with a single episode.18 Thus, the association

64 between primary HHV-6B infection and FS or febrile status epilepticus has been actively

65 studied, but the correlation is still controversial.19,20 Therefore, we compared the clinical

66 features and backgrounds of CFS patients with and without primary HHV-6B infection.

5
67 Methods

68 Study population

69 From October 2014 to September 2017, we enrolled 906 febrile children under age 5

70 who visited the emergency room or outpatient pediatric clinic of our university hospital, and

71 from whom blood was obtained for routine examination. Our facility takes care of all

72 children from primary to tertiary care. Among these patients, 62 patients who had no

73 convulsive disease in the past and were diagnosed with the first CFS were enrolled in this

74 study. For the purposes of our study, the case definition of a FS was a convulsive seizure in

75 children who had a fever above 38.0°C and no underlying disease such as central nervous

76 system infection, metabolic abnormality, or intoxication. Moreover, CFS was defined as a

77 focal or prolonged (>15 minutes) convulsion or as repeated convulsions within 24 hours.

78 Serum samples were collected at the time of admission to the hospital. Informed consent was

79 obtained from patient guardians after the project had been thoroughly explained. This study

80 was approved by the Ethical Review Board of Human Studies at Fujita Health University

81 (HM14-170).

82 Primary HHV-6B infection

83 Antibody titers to HHV-6 were measured by an indirect immunofluorescence assay,

84 as described previously.21 DNA was extracted from 200 µL of serum using a QIAamp Blood

85 Kit (QIAGEN, Chatsworth, CA, USA) and eluted in 50 μL of elution buffer, then stored at

6
86 −20°C. The details of the real-time PCR methods for measuring HHV-6B DNA copy

87 numbers have been described elsewhere.22 Primary HHV-6B infection was confirmed if the

88 following criteria were met: (1) HHV-6B DNA detection in serum obtained during the acute

89 phase of infection and (2) negative serum titers of antibodies to HHV-6B.

90 Data collections

91 We evaluated patient backgrounds such as age, gender and family history of FS and

92 clinical features of seizures such as type, duration, number within 24 hours after fever onset,

93 and interval between fever onset and seizure.

94 Statistical analyses

95 A statistical comparison between the two groups was performed by the chi-square

96 test and the Mann–Whitney U test. Kaplan–Meier plots were used to compare the recurrence

97 rate of subsequent FS between the two groups. Values of P<0.05 were considered to be

98 significant. The statistical analysis was performed with JMP version 13.2.1 (SAS Institute,

99 Cary, NC, USA).

7
100 Results

101 A total of 906 febrile children were enrolled during the observation period.

102 Sixty-two children (39 male and 23 female) were diagnosed with first-time CFS. The median

103 patient age was 16 months (range, 7–59 months). Thirty (48%) of the 62 patients were

104 diagnosed with primary HHV-6B infection. Patients’ backgrounds and clinical features were

105 compared between those with and without primary HHV-6B infection (Table 1). Those with

106 primary HHV-6B infection (median, 13 months; range, 7–39 months) were significantly

107 younger than those without primary HHV-6B infection (median, 19 months; range, 10–59

108 months) (P=0.001), and the proportion of males was significantly higher in patients without

109 primary HHV-6B infection (male/female, 25/7) than in those with the infection (male/female,

110 14/16) (P=0.017). There was no statistically significant difference in family history of FS

111 (P=0.569). No statistical differences were demonstrated in patient’s number of focal seizure

112 (P=1.000), seizure duration (P=0.37), and number of repeated FSs within 24 hours (P=0.339).

113 An interval between fever onset and seizures more than 24 hours after fever onset were

114 significantly more common in the patients with primary HHV-6B infection (15 (50%) of the

115 30 patients) than in those without primary HHV-6B infection (2 (6.3%) of the 32 patients) (P

116 <0.001).

117 The recurrence rates of FS after the first episode of CFS are shown by the Kaplan–

118 Meier curves in Figure 1. During the follow-up period of approximately 2.5 years, the

8
119 recurrence rate of FS was higher in patients with primary HHV-6B infection than in those

120 without viral infection, but this difference was not statistically significant (P=0.818).

9
121 Discussion

122 In this study, primary HHV-6B infection was identified in 32 (48%) of 62 CFS

123 patients. A recent meta-analysis of the association between HHV-6 and seizure demonstrated

124 that the incidence of HHV-6 infection in FS was 20% (119/592).23 The highest incidence was

125 43% (15/35), reported in a case-control study conducted in Canada in 1998.19 Additionally,

126 the latest observational study of FS performed in Australia demonstrated that the most

127 commonly identified viruses in FS patients were rhinoviruses, followed by adenovirus,

128 enteroviruses, influenza, and HHV-6 (HHV-6: 12%, 17/143).24 Thus, the incidence of

129 HHV-6B infection in this study was higher than that in all previous studies. It has been

130 demonstrated that FS associated with HHV-6 infection10,17 and influenza virus infection24,25

131 tends to be the complex type of FS. In contrast to previous studies, this study examined only

132 CFS patients, which is one of the major reasons for the high incidence of HHV-6B–associated

133 FS in this study. Meanwhile, it is considered that various infectious diseases patients were

134 included in the non-HHV-6 subjects. Therefore, in order to clarify clinical features of CFS

135 caused by each infectious disease, analysis of a large number of CFS patients combined with

136 diagnostic methods for more infectious agents is necessary in future studies.

137 Previous small cases studies carried out in Western countries demonstrated no

138 significant age difference between FS patients with and without HHV-6 infection.26 However,

139 our previous study, which examined 105 FS patients (including CFS patients), demonstrated

10
140 that FS patients with primary HHV-6 infection were significantly younger than those without

141 the viral infection.17 In this study, patients with primary HHV-6B infection were significantly

142 younger than those without (P=0.001). This suggests that CFS in younger children is more

143 commonly associated with HHV-6B than with other etiologic agents.

144 Our findings showed that the male/female ratio was significantly different between

145 patients with and without primary HHV-6B infection (P=0.02). As shown in the patients

146 without primary HHV-6B infection in this study, it was previously found that FS occurred

147 more frequently in males than females in Asia, including Japan, at a ratio of approximately

148 1.5:1.27,28 Meanwhile, in a population-based study of primary HHV-6 infection, Zerr et al.

149 demonstrated that the acquisition of HHV-6 was more common in females,29 but another

150 study carried out in Japan showed no gender difference in HHV-6B infection.17 Therefore,

151 these data suggest that HHV-6B–associated CFS, in which there was no gender difference is

152 caused by a different mechanism than CFS induced by other etiologies.

153 In this study, the frequency of three clinical features of CFS, namely focal seizures,

154 long-lasting seizures, and repeated seizures within 24 hours, were not different between

155 patients with and without primary HHV-6B infection. However, the number of patients who

156 had seizures more than 24 hours after fever onset was significantly greater in patients with

157 primary HHV-6B infection than in those without (P <0.001). In contrast to the present study,

158 the FEBSTAT study, which is an ongoing prospective multicenter study designed to elucidate

11
159 the relationship between febrile status epilepticus and subsequent hippocampal sclerosis and

160 mesial temporal lobe epilepsy30-32 demonstrated that the interval between fever onset and

161 seizure was not significantly different between patients with and without HHV-6 and -7

162 infection.20 However, that study analyzed only patients with febrile status epilepticus, which

163 is different from our study targeting CFS. A shorter interval between fever onset and FS has

164 been suggested to increase the risk of seizure recurrence.33 In the present study, although half

165 of patients with primary HHV-6B infection had CFS after longer than 24 hours from fever

166 onset, which is a reduced risk of recurrent FS, there was no significant difference in the

167 recurrence rate of FS between patients with and without primary HHV-6B infection. From a

168 clinical point of view, long intervals of more than 24 hours between fever onset and FS may

169 be useful in suggesting the presence of HHV-6B–associated FS, especially in infants and

170 young children.

171 Although this study demonstrated that HHV-6–associated CFS was not high risk of

172 recurrent FS, previous study has suggested that HHV-6B–associated FS increases in the risk

173 of subsequent epilepsy.17 Our previous study also demonstrated that HHV-6B DNA was

174 detected significantly more frequently in hippocampal brain tissue with mesial temporal

175 sclerosis than in brain tissue of non-mesial temporal sclerosis.34 Therefore, we hypothesize

176 that HHV-6B may play an important role in the pathogenesis of mesial temporal sclerosis,

177 through the alteration of host gene expression triggered by repetitive FS caused by HHV-6B.

12
178 If this hypothesis is correct, antiviral therapy may be considered for FS patient caused by

179 primary HHV-6B infection to prevent mesial temporal sclerosis. Although the small number

180 of cases and short observation period are major limitations of this study, the recurrence of FS

181 was higher in patients with primary HHV-6B infection than in those without it; however, this

182 difference was not significant (P=0.818). In order to determine whether or not HHV-6B

183 infection increases the risk of FS recurrence, it is necessary to follow a larger number of cases

184 for a longer period of time in a future study.

13
185 Acknowledgements

186 We thank Akiko Yoshikawa, Yoko Osakabe, and Chieko Mori for supporting our laboratory

187 experiments.

14
188 References

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19
269 Figure Legend

270 Figure 1. Cumulative recurrence rates of febrile seizures after the first complex febrile

271 seizure. The incidence of repeated febrile seizures after first episode of complex febrile

272 seizure was compared between patients with and without primary HHV-6B infection.

20
TABLE 1. Patient characteristics and clinical features

Primary No primary P value

HHV-6B infection HHV-6B infection
n=30 n=32

Age in months,
median (IQR) 13 (7–39) 19 (10–59) 0.0014
Sex, n
Male 14 25 0.0173
Female 16 7
Family history*
Yes 11 8 0.5694
No 16 19
Type of seizure
Focal 3 4 1.0000
Prolonged
≥ 15 minutes 5 9 0.3672
≥ 30 minutes 3 3 1.0000
Repeated within 24 h 26 24 0.3391
Fever-to-seizure interval
≥ 24 h 15 2 0.0001

* Information missing for 3 patients with primary HHV-6B infection and 5 patients with no primary
HHV-6B infection.
IQR; interquartile range, h; hour