Adenovirus infection associated with central nervous system dysfunction in children

Journal of Clinical Virology 57 (2013) 300–304

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Adenovirus infection associated with central nervous system dysfunction in children Yhu-Chering Huang a,b,∗,d , Sun-Lin Huang a,d,e , Shih-Perng Chen a , Ya-Ling Huang b,c,f , Chung-Guei Huang b,c , Kuo-Chien Tsao b,c , Tzou-Yien Lin a,b a

Department of Pediatrics, Chang Gung Memorial Hospital, Taoyuan, Taiwan Chang Gung University College of Medicine, Taoyuan, Taiwan c Department of Laboratory Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan b

a r t i c l e

i n f o

Article history: Received 19 December 2012 Received in revised form 12 March 2013 Accepted 26 March 2013 Keywords: Adenovirus Central nervous system dysfunction Seizure Encephalitis Children

a b s t r a c t Background: Adenovirus is a common pathogen in the pediatric population. No reports systemically evaluated central nervous system (CNS) dysfunction associated with adenovirus. Objective: To describe the detailed clinical features of adenovirus-associated CNS dysfunction in children. Study design: Of 3298 children with a throat virus culture positive for adenovirus treated at a university-affiliated hospital, from January 2000 to June 2008, in northern Taiwan, medical charts were retrospectively reviewed and those with CNS dysfunction were included. Results: 109 (3.3%) children had signs or symptoms of CNS dysfunction on admission. The median age was 3.6 years and 81 (76%) were less than 5 years of age. 64 (59%) were male. The most frequently cited CNS symptoms were seizure (64%), altered state of consciousness (13%), visual hallucination (9.3%) and lethargy (7.5%). The most common diagnoses included febrile seizure (48%), encephalitis (26%), afebrile seizure (11%) and meningitis/meningismus (8.3%). 31 (46%) of 67 patients receiving electroencephalogram examination had abnormal findings and 13 (36%) of 36 with image studies had abnormal findings. Ninety-nine (91%) children returned to normal health, 7 (6.4%) had a sequel of seizure disorder, and 3 (2.7%) had major sequelae and none died. Nine serotypes were identified, with three major serotypes (types 3, 2 and 1). Conclusion: CNS dysfunction was identified in 3.3% of children with adenoviral infection and most occurred in those <5 years old. The clinical prognosis seemed well. However, CNS dysfunction is a potentially serious complication of adenovirus infection in children. © 2013 Elsevier B.V. All rights reserved.

1. Background Adenoviruses are common pathogens, which are responsible for a varied array of illness in children, and are associated most commonly with respiratory illness and gastroenteritis, but ocular, cutaneous and urinary manifestations also occur frequently.1,2 Adenovirus infections can occur sporadically or in epidemics and can be identified throughout the year. At least 51 serotypes were identified and different serotypes may be implicated in different disease spectrum.1,2 For example, nasopharyngitis and tonsillitis

∗ Corresponding author at: Division of Pediatric Infectious Diseases, Chang Gung Memorial Hospital, No. 5, Fu-Shin Street, Kweishan, Taoyuan, Taiwan. Tel.: +886 3 3281200x8202; fax: +886 3 3288597. E-mail address: [email protected] (Y.-C. Huang). d These authors equally contributed to this manuscript. e Now in Lo-Tung St. Mary’s Hospital, Lo-Tung, Taiwan. f Now in E-Da Hospital, Kaohsiung, Taiwan. 1386-6532/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2013.03.017

are commonly associated with serotypes 1, 2, 3, 5 and 7; pharyngoconjunctival fever is usually caused by serotypes 2, 3, 4 and 7; pneumonia is usually caused by serotypes 3, 7 and 21; acute gastroenteritis is commonly caused by serotypes 40 and 41; and acute hemorrhagic cystitis is commonly caused by serotypes 7, 11 and 21.2 Although uncommon, adenovirus infection may present with central nervous system (CNS) dysfunction, and the presentation of CNS dysfunction may be associated with a disseminated disease at other body sites.1–4 A variety of clinical manifestations of CNS dysfunction have been observed and both meningitis and encephalitis have been noted as the major manifestations.1,2 Certain serotypes of adenoviruses have been reported to be implicated in CNS dysfunction1–3 and the virus or DNA can be identified from brain tissues or cerebrospinal fluid (CSF).1,2,5–7 Most of these patients only had mild CNS manifestations and may resolve without further complications. However, the reports on this issue were limited to case reports or case series of small numbers.5–17

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2. Objectives To describe the epidemiology, clinical manifestations and outcomes of adenovirus-associated CNS dysfunction in children.

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daily for cytopathic effects and identified using adenovirus specific monoclonal antibody (Chemicon, CA) by immunofluoresent assay.20,21 3.4. Genotyping

3. Study design 3.1. Case enrollment Patients less than 18 years of age with virus culture confirmed adenovirus infection were identified from the virology laboratory database of Chang Gung Memorial Hospital (CGMH), that provides from primary to tertiary care, situated in northern Taiwan, between January 2000 and June 2008. Of the patients with culture-confirmed (nasopharyngeal or throat swab) adenoviral infection, only those admitted to CGMH with signs or symptoms of CNS dysfunction were included in this study. The medical records were reviewed and the following data were collected: the demographics, underlying diseases, laboratory data (hemogram, CRP, CSF analyses, viral culture and blood culture if done), clinical presentations, diagnosis, electroencephalogram (EEG), image studies (including brain ultrasound examination, computerized tomography (CT) and magnetic resonance image (MRI), if done) and clinical outcomes. In our hospital, whether a lumbar puncture and image studies would be performed for a pediatric patient depended on the attending physician’s own discretion and a written informed consent should be obtained from the guardians. Usually, a patient with suspicious CNS infection and without symptoms/signs of severe increased intracranial pressure, a lumbar puncture would be suggested. We classified the outcomes into either returning to normal health (without neurological sequelae), recovery but with minor neurologic sequelae (e.g. persistent seizure disorder), recovery but with major sequelae (e.g. obstructive hydrocephalus, hemiplegia, vegetative state), or death.18

All the adenovirus isolates, if available, were typed by sequencebased method. Aliquots 200 ␮L of each of isolates known to be adenovirus culture positive from the clinical virology laboratory were retrieved from −80 ◦ C storage. Briefly, viral DNA was extracted from 200 ␮L of the adenovirus-positive viral isolates in a final elution volume of 60 ␮L using the Labturbo Virus Mini Kit (Taigen Bioscience Corp., Taipei, Taiwan). An initial volume of 5 ␮L of eluted sample was amplified using a PCR procedure that targeted hyper variable regions 1–6 of the hexon gene. M13 universal priming tails were added to the primers to facilitate sequencing.22 Expected amplicons from this reaction ranged from 764 to 896 bp in length. These amplicons were then sequenced on the ABI PRISM® 3100 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA). The forward and reverse sequences were generated from the assembly using the programs SeqMan in the Lasegene software package (DNASTAR, Madison, WI, USA). The database was available in GenBank using a BLAST. The serotype of each virus isolate to be identified was determined by the serotype of the highest scoring strain in the database, which is the sequence of the adenovirus strain that gave the highest nucleotide similarity value with the query sequence. 3.5. Statistics Data were analyzed by the SPSS program. Student’s t-test was used for continuous variables and Chi square test were used for categorical data. A p value less then 0.05 was denoted to be statistically significant.

3.2. Definitions

4. Results

Encephalitis was defined as patients who had consciousness change lasting >24 h and had either neurologic symptoms such as seizure and hallucination or abnormal EEG findings and occurring within 5 days of onset of an acute febrile respiratory illness that was proven subsequently to be adenovirus infection. Post-infectious encephalopathy was defined as patients who had consciousness change lasting >24 h and starting more than 5 days after the onset of respiratory symptoms.19 Aseptic meningitis was defined as patients with classic symptoms (fever, stiff neck, headache, vomiting) and pleocytosis in CSF (WBC ≥ 10 cells/␮L). For those with classic symptoms but without CSF samplings, they were categorized as meningismus.

4.1. Demographics and underlying diseases

3.3. Virology In our hospital, whether virus isolation and identification would be performed for a patient depended on the attending physician’s own discretion. Usually, the virus culture would be performed for a patient with presumed viral infection if the attending doctor would like to uncover the possible viral pathogen. Specimens were obtained from throat or nasopharyngeal swabs, collected and transported in a viral transport media to the virologic laboratory within four hours. Viral isolation was carried out by tissue cultures. Respiratory specimen was inoculated into four different cell lines, including human epidermoid carcinoma cell (Hep-2), canine kidney cell (MDCK), human embryonal lung cell (MRC-5), and rhesus monkey kidney cell (MK-2). Cultures were maintained in minimal essential media containing antibiotics and incubated at 33 ◦ C, rotated at 12 revolutions per hour. All cultures were observed

We identified 3298 patients with adenovirus infection at CGMH during the study period. 109 of the 3298 patients (3.3%) had signs or symptoms of CNS manifestations at the time of admission. The median and mean age was 3.61 years and 3.62 years (±2.59), respectively, ranging from 0.3 to 12.4 years and 83 children (76.1%) were aged ≤ 5 years. The ratio of males to females was 1.42 (64 vs. 45 patients). Ten children had a history of febrile convulsion, four patients had a history of seizure disorder, and five children were premature born babies (one had a history of intraventricular hemorrhage with resultant periventricular leukomalacia (PVL)). The median hospitalization stay was 6 days, ranging from 2 to 399 days. Four patients (3.7%) had a hospital stay more than 30 days. 4.2. Clinical symptoms/signs and clinical diagnosis Regarding clinical manifestations, 103 patients (94.5%) had fever, 67 (61.5%) had rhinorrhea, 65 (59.6%) had cough, 61 (56%) had tonsillitis, and 34 (31.2%) had vomiting. The most frequently cited CNS symptoms were seizure (62.4%), altered state of consciousness (12.8%), headache (11.9%), visual hallucination (9.2%) and lethargy (7.3%) (Table 1). Laboratory data of the 109 children are shown in Table 2. 31 patients had leukocytosis and 28 patients had a serum C-reactive protein level ≥40 mg/L (normal, <5 mg/L). Of the 109 patients, febrile seizure was the most common clinical diagnosis and accounted for 52 patients (48%). All the 10 children with a history of febrile convulsion presented with febrile seizure, while of the four patients with a history of seizure disorder, two

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Table 1 Presenting symptoms or signs of central nervous system (CNS) dysfunction in 109 children with adenoviral infection. CNS symptoms or signs

No. (%)

Seizure Altered state of consciousness Headache Visual hallucination Lethargy Limbs paralysis Neck stiffness Myoclonic jerk

68 (62) 14 (13) 13 (12) 10 (9.2) 8 (7.3) 6 (5.5) 2 (1.8) 2 (1.8)

months, hemiplegia with subsequent limbs atrophy, respectively; two presented with encephalitis) and none died. Four patients (3.7%) had a hospital stay more than 30 days and three of them had a diagnosis of encephalitis and the other ADEM. Three of them (75%) had a major sequel. 4.5. Adenovirus genotyping Of the 109 cases, 99 isolates were available for serotyping analysis (Table 2). A total of nine genotypes were identified. The most common genotype was type 3 (44.2%), followed by types 2 (17%) and 1 (13%). No significant association was identified between genotypes and clinical disease entities.

presented febrile seizure and afebrile seizure, respectively, at this time. Two patients had two diagnoses for CNS dysfunction and were aseptic meningitis plus acute flaccid paralysis and seizure, respectively. 28 patients (28%) had encephalitis, three had meningoencephalitis, 12 (11%) had afebrile seizure, and nine (8.3%) had aseptic meningitis/meningismus (Table 3).

5. Discussion Although there have been reports regarding CNS dysfunction implicated in patients with adenovirus infection, most reports were limited to case reports or case series of small case numbers.5–17 To our knowledge, this is the first report which systemically evaluated CNS dysfunction associated with adenoviral infection in children. Results from the present study showed that 3.3% of 3298 children with adenovirus infection had the manifestation of CNS dysfunction. The incidence rate of CNS dysfunction associated with adenoviral infection in the present study may be overestimated since nearly half of the patients presented with febrile seizure. If we excluded the cases of simple febrile seizure, the incidence rate would be reduced to 1.7%. However, Simila et al.13 reported that 8 of 32 infants and children with adenovirus type 7 infection in an epidemic in Finland had a manifestation of meningitis or meningoencephalitis, a rate of CNS involvement even higher than that in the present study. In the present study, the most common presenting signs or symptoms of CNS dysfunction associated with adenoviral infection were seizure, altered state of consciousness, headache, and visual hallucinations and the most common diagnoses were febrile seizure, encephalitis/encephalopathy, afebrile seizure and aseptic meningitis/meningismus. All these observations were consistent with previous reports.1,2 In addition to meningitis or meningoencephalitis, Straussberg12 reported seven children infected with adenovirus type 3 initially had typical manifestations of adenoviral infection, such as pneumonia, tonsillitis, and later developed transient encephalopathy. Cerebrospinal fluid evaluations were within normal limits in all patients and neurologic findings resolved in about one week, which were consistent with those in the present study. Several investigators have demonstrated the utility of PCR detection of adenoviral DNA in acute respiratory infections in immunocompetent patients.2 Quantitative real-time PCR assay would increase sensitivity and even a multiplex PCR assay for

4.3. Image and laboratory data Sixty-seven patients received EEG examination. 31 patients (46%) had abnormal findings, including focal or diffuse cortical dysfunction for 12 patients, focal or multi-focal epileptiform discharge for 9 patients and both cortical dysfunction plus epileptiform discharge for 10 patients. 38 patients had image studies, which included brain CT scan for 21 children, MRI for 5, both CT and MRI for 4 and ultrasound for 8, and 13 patients (36%) had abnormal findings, including subdural effusion for 5 patients, brain swelling, increased intracranial pressure, and brain atrophy, respectively, for two each and hydrocephalus and PVL for one each. Lumbar puncture was performed in 28 children. 6 of 23 patients (26%) showed pleocytosis and 4 of 27 patients (14.8%) showed elevated CSF protein. Adenovirus was not detected in any case by CSF viral culture and the CSF bacteria culture all showed no growth (Table 4). 4.4. Clinical outcomes Two patients, one with a history of PVL, presented with status epilepticus and both, thereafter, had a sequel of seizure disorder requiring long-term anticonvulsant therapy. The patient presenting with acute disseminated encephalomyelitis (ADEM) developed obstructive hydrocephalus and a ventriculoperitoneal (V-P) shunt was inserted subsequently. One patient presenting with febrile seizure also had hepatitis, and thereafter, had a sequel of seizure disorder. Totally, 99 children (91%) returned to normal health, 7 (6.4%) recovered with minor neurologic sequelae (seizure disorder in all cases), 3 (2.7%) recovered with major neurologic sequelae (one each with vegetative status, hydrocephalus post status of V-P shunt insertion and transient ataxia and dysmetria for 2

Table 2 Distribution and correlation of adenovirus serotype and clinical diagnoses in 109 patients with central nervous system dysfunction. Serotype

1

2

3

4

5

6

7

11

UT

41

NA

Total

Number Febrile seizure Encephalitis Meningitis/meningismus Meningo-encephalitis Afebrile seizure Status epilepticus Acute flaccid paralysis Cerebellitis ADEM Extrapyramidal syndrome

14 (13) 7 3 2a 0 0 1 1a 1 0 0

19 (17) 12 3 0 0 4 0 0 0 0 0

47 (43) 23 16 3 1 2 1 0 0 0 1

5 (4.6) 2 2 1 0 0 0 0 0 0 0

4 (3.7) 2 1 0 0 1 0 0 0 0 0

2 (1.8) 1 1 0 0 0 0 0 0 0 0

2 (1.8) 1 0 0 0 1 0 0 0 0 0

1 (0.9) 0 0 1 0 0 0 0 0 0 0

3 (2.8) 2 0 0 0 0 0 1 0 0 0

2 (2.3) 0 0 0 1 0 0 0 0 1 0

10 (9.2) 2 2 2a 1 4a 0 0 0 0 0

109 52 (48) 28 (26) 9 (8.3) 3 (2.8) 12 (11) 2 (1.8) 2 (1.8) 1 (0.9) 1 (0.9) 1 (0.9)

UT, undetermined; NA, not available; ADEM, acute disseminated encephalomyelitis. a A single patient with two diagnoses was noted in two cases.

Y.-C. Huang et al. / Journal of Clinical Virology 57 (2013) 300–304 Table 3 Laboratory data of 109 children with adenoviral infection and central nervous system dysfunction. Laboratory data Leukocyte count (/mm3 )a Median Mean ± standard deviation Leukocytosis (≥15,000/mm3 ) C-reactive protein (mg/L) (normal, <5 mg/L)b Median Mean ± standard deviation ≥40 mg/L ≥100 mg/L Cerebrospinal fluid analysisc Pleocytosis (leukocyte count ≥ 10 cells/␮L) Elevated protein (>45 mg/dL) Positive culture for bacteria Positive culture for virus a b c

No. (%) or measure value 11,700 12,731 ± 5230 31 (28) 18 36 ± 44 30 (28) 10 (9.2) 6/23 (26) 4/27 (16) 0/28 (0) 0/17 (0)

Only 106 cases had leukocyte data. Only 105 cases had C-reactive protein data. Only 28 patients had lumbar puncture examination.

rapid, type-specific identification of adenovirus serotypes had been developed. Other investigators have demonstrated the utility of using quantitative real-time PCR assays to diagnose and monitor adenoviral infection and disease in immunocompromised hosts.2 PCR should be performed on the nucleic acid isolated from the CSF and did identify several cases of encephalitis due to adenovirus in the literature.23 However, though PCR are the tests of choice for many viral agents of encephalitis and meningitis, certain viruses such as influenza viruses, poliovirus and enterovirus 71, which are well documented to be implicated in either meningitis or encephalitis, are not frequently identified from CSF by either virus culture or PCR. The sensitivity and specificity of PCR for the detection of adenovirus in the CSF specimens needs further studies. In our hospital, PCR for the detection of adenovirus is not included for those with CNS infections. As the serotypes implicated in CNS dysfunction, adenovirus types 1, 2, 3, 5, 6, 7, 12, 26 and 32, in isolated instances, have been recovered from both brain and CSF. In the present study, type 3 was the most common type identified and most other types identified were among the previously reported types implicated in CNS dysfunction. However, types 4, 11 and 41 were not reported before. Based on biochemical and biophysical criteria,1,2 adenovirus type 4 was categorized to subgroup E, type 41 to subgroup F and type 11 to subgroup B2; in contrast, types 1, 2, 5, 6 were categorized to subgroup C, types 3 and 7 to subgroup B1, type 12 to subgroup A and type 32 to subgroup D. Different subgroups with different biochemical and biophysical characteristics may be implicated in different manifestations, such as respiratory tract, eyes, gastrointestinal tract or urological tract. However, there have been inadequate data to suggest the association between adenovirus subgroups and neurologic manifestations. Table 4 Electroencephalography (EEG) and brain image studies in the children with adenoviral infection and central nervous system dysfunction. Examinations

No. (%)

Abnormal EEG findings Focal or diffuse cortical dysfunction Focal or multifocal epileptiform discharge Both Abnormal image studies Subdural effusion Brain swelling or increased intracranial pressure Brain atrophy Hydrocephalus Periventricular leukomalacia

31/67 (46) 12 9 10 13/36 (36) 5 4 2 1 1

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The outcome of the patients with CNS dysfunction associated with adenoviral infection were usually well, though the case number was small,1,2 and most patients returned to normal health without neurological sequelae, as the patients in the present study, in which more than 90% of the patients recovered uneventfully. Though the incidence rate of CNS dysfunction associated with adenoviral infection was low and the outcome was usually not bad, it is still a potentially serious complication since almost every child would have at least one episode of adenovirus infection before 5 years old.1,2 Since this is a retrospective study, there are several inherent limitations in the present study. CSF sampling was not obtained from each patient and adenovirus was not identified from any CSF sample. Though the patient, from whom other etiologic microbiologic agent was identified, was excluded for analysis, not every patient received a complete and consistent survey for all possible pathogens implicated in CNS involvement. Likewise, though the virus or DNA could be identified from brain tissues or CSF in some cases previously,1,2,5–7 most reported cases lacked direct virologic evidence as the present study. Instead of virus isolation, polymerase chain reaction may be a useful diagnostic tool to detect adenovirus DNA in the CSF and should be included and evaluated in the future study. In addition, not every patient had image studies and EEG examination. All these factors might affect the incidence rate determination of each manifestation and the clinical perspective of CNS dysfunction associated with adenoviral infection. In conclusion, adenovirus infection with CNS involvement in children was uncommon and if occurred, most of them were children aged < 5 years. The most common diagnosis was seizure, either febrile or afebrile, followed by encephalitis. The clinical prognosis of these cases seemed well. However, we cannot disregard this issue since adenovirus infection is very common in children. Funding This study was supported by a grant from Chang Gung Memorial Hospital (CMRPG 470131). Competing interests None. Ethical approval This study was approved by the Institutional Review Board of Chang Gung Memorial Hospital. References 1. Williams JV. Adenoviruses. In: Kliegman RM, Stanton BF, Geme III St, Schor JW, Behrman NFRE, editors. Nelson textbook of pediatrics. 19th ed. Philadelphia, PA: WB Saunders, Elsevier; 2011. p. 1131–3. 2. Cherry JD, Chen TK. Adenovirus. In: Feigin, Cherry, editors. Textbook of pediatric infectious diseases. 6th ed. Philadelphia, PA: WB Saunders; 2009. p. 1949–72. 3. Schmltz H, Wlgand R, Helnrich W. Worldwide epidemiology of human adenovirus infections. Am J Epidemiol 1983;117:455–66. 4. Hong JY, Lee HJ, Piedra PA, Choi EH, Park KH, Kho YY, et al. Lower respiratory tract infections due to adenovirus in hospitalized Korean children: epidemiology, clinical features, and prognosis. Clin Infect Dis 2001;32:1423–9. 5. Dubberke ER, Tu B, Rivet DJ, Storch GA, Apisamthanarak A, Schmidt RE, et al. Acute meningoencephalitis caused by adenovirus serotype 26. J Neurovirol 2006;12:235–40. 6. Chartterjee NK, Samsonoff WA, Balasubramaniam N, Rush-Wilson K, Spargo W, Church TM. Isolation and characterization of adenovirus 5 from the brain of an infant with fatal cerebral edema. Clin Infect Dis 2000;31:830–3. 7. Frange P, de Latour RP, Arnaud C, Boddaert N, Oualha M, Avettand-Fenoel V, et al. Adenoviral infection presenting as an isolated central nervous system disease without detectable viremia in two children after stem cell transplantation. J Clin Microbiol 2011;49:2361–4. 8. Faulkner R, Van Rooyen CE. Adenovirus types 3 and 5 isolated from the cerebrospinal fluid of children. Can Med Assoc J 1962;87:1123–5.

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