Jpn. J. Infect. Dis., 61, 18-24, 2008

Original Article Clinical Application of Reverse-Transcription Polymerase Chain Reaction and Intravenous Immunoglobulin for Encephalitis Ming-Fang Cheng1,4, Bao-Chen Chen2,4, Tsi-Shu Huang2,4, Kai-Sheng Hsieh1,4, Shu-Nuan Chen2,4 and Yung-Ching Liu2,3,4* 1Department of Pediatrics, 2Department of Microbiology, and 3Section of Infectious Diseases, Department of Internal Medicine, Veterans General Hospital-Kaohsiung, Kaohsiung, and 4National Yang-Ming University, Taipei, Taiwan (Received June 5, 2007. Accepted October 19, 2007) SUMMARY: Although polymerase chain reaction (PCR) is a highly sensitive procedure for the diagnosis of , it has never been systemically applied to the treatment of enteroviral encephalitis using intravenous immunoglobulin (IVIg). We conducted a 2-year randomized, controlled comparison of reverse transcription (RT)-PCR of cerebrospinal fluid (CSF) with traditional viral isolation to guide IVIg treatment. Seventy-five patients were enrolled and classified into three groups: one group with clinical manifestations of enteroviral and two without. The latter two groups were separated on the basis of whether IVIg treatment was guided by RT-PCR or virus culture assay. CSF specimens from the 18 confirmed cases of enteroviral encephalitis were RT-PCR positive for enterovirus in all but one case. Of the remaining 57 cases of nonenteroviral encephalitis, only 4 were positive for enterovirus RT-PCR. One patient in the group of IVIg treatment guided by viral isola- tion subsequently displayed a sequel of epilepsy. No patients in the IVIg treatment groups guided by RT-PCR had any neurological sequelae. In conclusion, the use of RT-PCR allowed rapid, sensitive, and specific detection of enteroviral RNA in CSF. When used to guide IVIg treatment, RT-PCR may shorten hospitalization and improve outcomes of patients with enteroviral encephalitis.

with the host’s age and immune status. Virulence determi- INTRODUCTION nants of the circulating virus may also be involved (9,10); An enterovirus (EV) epidemic in Taiwan in 1998, predomi- antibodies play a pivotal role in resolving . Indeed, nantly caused by EV 71 in 42% of cases, coxsackievirus the use of immune globulin has quelled the incidence of A16 in 18%, and other EVs in 40%, resulted in a total of enteroviral infections in the nursery (11-13) and in individu- 129,106 cases reported to have the primary clinical marker ally ill newborns (14-16). However, the clinical efficacy of of enteroviral infection-- and hand-foot-mouth- IVIg in enteroviral encephalitis remains controversial (9). disease (HFMD), with 405 severe cases and 78 deaths (1-3). Patients with encephalitis who present characteristic This epidemic prompted the use of intravenous immuno- herpangina or HFMD are easily diagnosed as enteroviral globulin (IVIg) for the treatment of severe enteroviral infec- infections. However, many patients with enteroviral encepha- tions, including EV-mediated encephalitis in Taiwan (4). This litis do not display these characteristic manifestations; instead, treatment policy has been hampered by the need to prove the diagnosis relies on the direct detection of the virus from presence of enteroviral infections for those patients without cerebrospinal fluid (CSF), throat swab, or stool. These detec- characteristic herpangina or HFMD, since current tissue tion methods suffer from a slow turnaround time (17) and culture-based methods are laborious, time-consuming, and relative insensitivity (17,18). Brain biopsy, perhaps the only frequently unsuccessful exercises (5). In contrast, detection existing “gold standard” in diagnosing , is of EVs by reverse transcription-polymerase chain reaction rarely justified because of its invasive nature. (RT-PCR) is more rapid and sensitive (6). The objective of RT-PCR more rapidly and sensitively detects EV genomic the present study was to assess the impact of high-dose IVIg RNA in specimens as varied as muscle biopsy, CSF, throat adjuvant therapy in patients with enteroviral encephalitis swabs, serum, and stool (18,19). We used a commercially avail- diagnosed by the RT-PCR assay. able PCR assay (AMPLICOR EV Test; Roche Molecular EVs are a leading cause of meningoencephalitis (7). They Systems, Branchburg, N.J., USA) that combined sufficient reside in a genus within the family of Picornaviridae, and sensitivity and maximum specificity (17,18,20) for the rapid are single-stranded RNA viruses that can be subdivided into detection of EV RNA in clinical specimens by taking advan- five species including human enterovirus (HEV) A to D tage of the new real-time LightCycler PCR technology (6) species and type species (8). Specific antiviral treat- using the TaqMan format (21). ment is not currently available. The clinical disease observed Presently, we conducted a detailed prospective study of during central nervous system enteroviral infections varies the clinical application of RT-PCR and the effects of treat- ment with IVIg on symptomatic enteroviral encephalitis. *Corresponding author: Mailing address: Section of Infectious Diseases, Department of Internal Medicine, Veterans General MATERIALS AND METHODS Hospital-Kaohsiung, 386 Ta-Chung 1st RD, Kaohsiung, Taiwan 81346. Tel: +886-7-3422121 ext. 2029, Fax: +886-7-3468067, Study design: The subjects (n = 75; all <15 years of age) E-mail: [email protected] enrolled in the prospective study were admitted to Veterans

18 General Hospital-Kaohsiung, Taiwan, presenting with indi- for an additional 14 days. Once a cytopathic effect involved cations of encephalitis from January 1, 2003 to December over 50% of the cell monolayer, the cells were scraped 31, 2004. The clinical criteria for encephalitis included loose and indirect fluorescent antibody staining with a pleocytosis (leukocytes ≥25/mm3), an absence of bacteria panenteroviral monoclonal antibody (Chemicon International, upon culture of CSF, and some or all of the following symp- Inc., Temecula, Calif., USA) was performed to identify the toms: fever; sensorium changes (such as confusion, drowsi- EV antigens. All isolates were subsequently serotyped by ness, stupor, coma, combativeness, convulsions, abnormal immunofluorescence with type-specific monoclonal anti- behavior, ataxia, limb paralysis, and clumsiness), hemiplegia, bodies (all from Chemicon International) to poliovirus 1 to 3; specific cranial nerve dysfunction, and brain dysautonomia. coxsackievirus A2, A6 to A16, A24, and B1 to B6; echovirus At the time of the study the use of IVIg therapy for enteroviral 3, 4, 6, 9, 11, and 30; EV 70; and EV 71. In addition, EV 71 meningitis was not prompted in Taiwan. Hence, patients VPI-IgM Rapid tests (Oncoprobe Biotech, Taipei, Taiwan) presenting with symptoms or signs of meningitis such as head- were carried out to identify the EV 71 infection for the serum ache, stiff neck, nausea, and vomiting, but without the defin- samples from all of the enrolled cases. able signs of encephalitis summarized above, were excluded. RNA extraction and reverse transcription: RNA was Encephalitis caused by nonviral pathogens cultured in CSF extracted from 140 μl of CSF or viral culture supernatant (such as bacteria, mycobacterium, fungus, parasites) and con- using the QIAamp viral RNA extraction kit (Qiagen, Hilden, scious changes caused by factors other than infection (such Germany) according to the manufacturer’s instructions. RNA as hemorrhage, infarction, hypoglycemia, or other metabolic was recovered in 60 μl of nuclease-free water and either disorders) were also excluded in this study. Standard labo- used immediately or stored at –80°C until further analysis. ratory analyses conducted upon admission included viral cDNAs were obtained by RT using the Reverse Transcrip- cultures of the throat, rectal or stool specimens, CSF, and tion System (Promega, Madison, Wis., USA). urine. LightCycler PCR: A master mix (LightCycler FastStart Among the enrolled encephalitis patients, those who had DNA Master Hybridization Probes; Roche Diagnostics characteristic manifestations of herpangina or HFMD were GmbH, Penzberg, Germany) was optimized for the defined as clinically diagnosed enteroviral encephalitis, and LightCycler. The mix was composed of 0.2 mM concentra- those who had positive cultures of nonpolio EVs were tions of each of the deoxyribonucleoside triphosphates (50 mM defined as virus-culture confirmed enteroviral encephalitis. KCl, 10 mM Tris-Cl [pH 8.3]), 3 mM MgCl2, 0.5 μM con- Patients who did not have positive cultures of EVs nor char- centrations of the primers (forward primer: 5´-GTA ACG GGC acteristic manifestations of herpangina or HFMD were defined AAC TCT GCA GC-3´; reverse primer: 5´-ATT GTC ACC as nonenteroviral encephalitis. ATA AGC AGC CA-3´), and 0.2 μM of the fluorescein probe The patients were divided into three groups. Clinically (5´-6FAM-CAD GGA CAC CCA AAG TAG TCG GTT CC diagnosed patients with enteroviral encephalitis were assigned TAMRA-TP-3´). Five microliters of cDNA was added to 15 to group A. The CSF of these patients was evaluated by RT- μl of PCR mixture in each reaction capillary. The reaction PCR within 24 h of admission, and IVIg (1 g/kg body weight; mixture was centrifuged in the capillary to facilitate mixing. the same dose was used for groups B and C) was adminis- All capillaries were then sealed and amplified using the tered via an intravenous route (groups B and C as well) after following protocol: 95°C for 2 min for one cycle, followed withdrawing the CSF sample. Patients with encephalitis with- by 5 s of denaturation at 95°C, 10 s of annealing at 65°C, and out herpangina or HFMD were randomized into groups B 10 s of primer extension at 72°C for 40 cycles. The melting and C. CSF specimens of the group B patients were sent for curve analysis was performed in one cycle of 95°C for 10 s RT-PCR within 24 h of admission, and IVIg was adminis- and 50°C for 60 s, each with a temperature transition rate tered to those patients upon a positive result. The CSF of the of 20°C/s, and then ramping to 85°C at 0.1°C/s. Negative group C patients was not analyzed by RT-PCR until near the controls without virus cDNA were included in every PCR. completion of the study. Additionally, group C patients were not administered a placebo, and IVIg was not administered RESULTS until the isolation of EV. All patients received standard sup- portive care as determined by their primary care providers. Seventy-five cases with aseptic encephalitis were enrolled With the exception of IVIg treatment, all treatment regimens during the 2-year study. Of these, 29 (39%) occurred in the and management decisions, including duration of hospitaliza- summer (between May and July); the seasonal distribution tion, were determined by the primary caregivers and were among the three groups was not significantly different (P = unaffected by the study protocol among the different groups. 0.43). Eighteen cases were caused by EVs, and the other 57 Patients were reevaluated during follow-up visits at 7 and 30 cases were nonenteroviral. days, 3 months, and every 3 months after discharge until the The serotype distribution and the specimens used to iso- final visit at the end of 2006. late EVs and nonenteroviral viruses in groups A, B, and Viral isolation and serotyping: CSF, throat swabs, urine, C during the entire course of the study are summarized in stools, and/or rectal swabs from the patients were used for Table 1. Of the 18 CSF specimens acquired from the patients virus isolation. Samples were inoculated onto RD, A549, LLC having virus-culture confirmed enteroviral encephalitis, 17 MK2, Vero, and HEL cells. A culture positive for nonpolio were EV positive by RT-PCR; the remaining specimen was EVs from any source was accepted as evidence of enteroviral found to contain coxsackievirus A9. Of the CSF specimens infection. Urine was added to Eagle’s minimum essential obtained from patients with nonenteroviral encephalitis, 4 medium (EMEM) and passed through a 0.2 μm pore size were EV-positive and 53 were EV-negative by RT-PCR. filter prior to the culture. Cultures were maintained in EMEM RT-PCR was performed within 24 h of admission for all with 2% FCS at 37°C in an atmosphere of 5% CO2 and were 10 patients in group A (i.e., patients with herpangina or monitored daily for cytopathic effects. Samples that did not HFMD) (Fig. 1). All CSF specimens from this group were display cytopathic effects on day 7 were passed and observed positive for EV by RT-PCR, and at least one specimen was

19 Table 1. Distribution of virus isolates and serotypes in the different groups

Groups/patients Results of CSF EV RT-PCR and culture from various specimens with culture Serotype1) RT-PCR Cultures confirmed encephalitis CSF CSF Throat swab Rectal swab Stool Urine Group A Patient 1 Echovirus 11 + + – NA + – Patient 2 Echovirus 11 + + + + NA + Patient 3 Echovirus 11 + – +NA–– Patient 4 Echovirus 11 + – ++NA– Patient 5 Echovirus 11 + – ++NA– Patient 6 Echovirus 9 + –– +NA– Patient 7 Echovirus 9 + + – NA + – Patient 8 Coxsackievirus A9 + – + – NA – Patient 9 Coxsackievirus B3 + –– +NA– Patient 10 Nontypable + – + – NA – Group B Patient 1 Echovirus 11 + + – +NA– Patient 2 Echovirus 11 + – +NA+– Patient 3 Coxsackievirus A9 + –– +NA– Patient 4 Nontypable + + + – NA – Patient 5 –– +NA– + Patient 6 Cytomegalovirus –– – – NA + Patient 7 Adenovirus + – + – NA – Patient 8 A virus –– +NA–– Group C Patient 12) Echovirus 11 + – + – NA – Patient 2 Echovirus 11 + – +NA+– Patient 3 Coxsackievirus A9 –– – NA + – Patient 4 Coxsackievirus B3 + + – +NA– Patient 5 Cytomegalovirus –– + – NA – Patient 6 Adenovirus –– + – NA – Patient 7 –– +NA–– Patient 8 HSV type 2 + + + ––– 1): EV-serotyping was done for all of the specimens culture-positive for virus. 2): The patient with symptoms of epilepsy in the post-discharge follow-up. CSF, cerebrospinal fluid; EV, enterovirus; HSV, virus; +, positive; –, negative; NA, not available. culture-positive for EV. The 10 isolates included echoviruses with echovirus 11 who did not receive IVIg until detection 11 and 9 (n = 5, 2, respectively), and coxsackieviruses A9 by viral isolation displayed symptoms of epilepsy at the post- (n = 1), and B3 (n = 1), and untypable EV (n = 1). All 10 discharge follow-up visit. The EV 71 VPI-IgM Rapid test to patients received IVIg soon after admission. The other detect serum EV 71 IgM and the results of indirect immuno- 65 patients without herpangina or HFMD were randomly fluorescence staining with monoclonal antibodies (mAbs) assigned to group B (RT-PCR conducted within 24 h of against EV 71 (mAb 979, 3323, and 3324) were negative in admission; n = 32) and group C (RT-PCR performed later in all 75 cases. the study; n = 33). Five group B patients were PCR-positive There were no significant differences between the three for EV and immediately received IVIg. Four of these 5 groups in baseline demographic, clinical, and laboratory char- patients were subsequently culture-positive for echovirus 11 acteristics (Table 2). Infusions of IVIg (1 g/kg body weight) (n = 2), coxsackievirus A9 (n = 1), and an untypable EV (n = in the 17 patients who were so-treated were generally well- 1). The 27 patients who were PCR-negative for EV were subse- tolerated. Skin , fever, tachycardia, and tachypnea were quently negative on viral culture. Of the group C patients, 4 observed in 2 patients; these potential but unconfirmed side were culture-positive for EV including echovirus 11 (n = 2), effects did not prompt discontinuation of the infusion. No coxsackievirus A9 (n = 1), and coxsackievirus B3 (n = 1). association of IVIg and clinically significant changes in renal The patients infected with coxsackievirus A9 were subse- function, neutropenia, or thrombocytopenia were observed quently PCR-negative for EV, and the other 3 patients culture- in the 2-year-long study. No fatalities occurred during the positive for EV were subsequently PCR-positive. The patient study among those with aseptic encephalitis. During post- infected with coxsackievirus B3 and one of the patients discharge follow-up, only a single group C patient infected infected with echovirus 11 had frequent seizure episodes; with echovirus 11 displayed neurological sequelae (epilepsy). IVIg was administered upon identification of the particular The two most common EV serotypes were echovirus 11 EV. The other 29 group C patients whose enteroviral isola- and 9, and the two most common nonenteroviruses were tion was negative did not receive IVIg during hospitaliza- cytomegalovirus and adenovirus. Bacterial cultures of blood, tion. However, 3 patients subsequently proved to be virus- CSF, and urine were negative for all of the 75 patients, positive in retrospective RT-PCR analysis. A patient infected except for 4 urine cultures subsequently determined to be the

20 Fig. 1. Flow chart of study enrollment and outcomes.

Table 2. Baseline characteristics of the patients in the different groups1) such as oseltamivir, acyclovir, and gancyclovir, respectively, Group A Group B Group C no matter which group they belonged to. Characteristic (n = 10) (n = 32) (n = 33) The median time to discharge was 10, 9, and 14 days in groups A, B, and C, respectively (P = 0.08). There was no Age (y) 1.62 ± 2.11 1.59 ± 3.25 1.62 ± 2.97 statistically significant difference in the duration of hospital- No. of male (%) 6 (60) 15 (47) 17 (52) ization between the three groups, though the patients in group White cell count per mm3 6,725 ± 4,126 7,510 ± 7,531 6,980 ± 5,968 C stayed in the hospital 4 and 5 days longer than those in C-reactive protein, mg/dL 2.3 ± 3.9 2.9 ± 4.6 2.7 ± 5.1 groups A and B, respectively. Blood sugar, mg/dL 92.5 ± 20.3 89.3 ± 45.2 96.2 ± 35.6 Cerebrospinal fluid White cell count per mm3 54 ± 126 46 ± 154 60 ± 127 DISCUSSION Red-cell count per mm3 8 ± 25 12 ± 35 10 ± 28 During the epidemic of enteroviral infection in 1998, the Glucose, mg/dL 73.2 ± 32.0 68.0 ± 28.0 65.9 ± 27.2 viruses isolated at two major diagnostic laboratories that Protein, mg/dL 40.0 ± 18.2 42.2 ± 20.9 35.7 ± 15.8 processed samples from inpatients and outpatients, suggested 1): Plus-minus values are mean ± SD. There were no significant differ- that although approximately half of the EVs isolated were ences among the groups. EV 71, other EVs were also active. Coxsackievirus A16 was known to cause 18% of the cases, and 40% of the cases were result of specimen contamination. All the patients with posi- caused by other EVs (1-3). After this severe epidemic, IVIg tive cultures of influenza A or B virus, , was introduced for the treatment of severe enteroviral infec- and cytomegalovirus were treated with specific antiviral drugs tions, including enteroviral encephalitis. The indications of

21 IVIg treatment are not only limited to severe EV 71 infection. The criteria for the indications include patients with clinical manifestations of HFMD or herpangina or epidemiological evidence of enteroviral infection, and the presence of at least one severe clinical syndrome (4). This policy for IVIg administration has been hampered by the need to prove the presence of enteroviral infections for those patients without characteristic herpangina or HFMD, since current tissue culture-based methods are laborious, time- consuming, and frequently unsuccessful exercises (5). Patients having enteroviral encephalitis without the characteristic Fig. 2. Distribution of serotypes in patients of virus-culture confirmed occurrences of herpangina or HFMD (8/18 patients, 44%, in enteroviral encephalitis from 1999 through 2006. the present study) are typically diagnosed by the isolation of the causative virus from clinical specimens. While conclu- present as contaminants within IVIg) (35). Wang et al. sive, this route of diagnosis is long and laborious, and so is demonstrated the modulation of cytokine production by IVIg entirely impractical for rapid diagnosis and prompt treatment. in patients with EV 71-associated brainstem encephalitis. In the present study, biochemistry and cell counts in CSF, white Plasma levels of inferferon (IFN)-γ, interleukin (IL)-6, cell counts, and serum C-reactive protein levels were indistin- IL-8, IL-10, and IL-13 levels significantly decreased in guishable between enteroviral encephalitis and nonenteroviral patients with pulmonary edema after administration of IVIg. encephalitis (Table 2). Thus, differentiation of enteroviral from Additionally, plasma levels of IL-6 and IL-8 were signifi- nonenteroviral encephalitis on the basis of hematology and cantly decreased in patients with autonomic nervous system biochemical analysis is virtually impossible. dysregulation after administration of IVIg. These findings sug- In the present study, the sensitivity, specificity, positive gest that IVIg may play a therapeutic role in EV 71-associated predictive value, and negative predictive value were 94% (17/ brainstem encephalitis and indicate the possible mechanisms 18), 93% (53/57), 81% (17/21), and 98% (53/54), respec- of IVIg therapy in such kinds of severe enteroviral infection tively. These values are very similar to other reported study (36). The role of IVIg in the treatment of viral encephalitis results (17,18,20), indicating the suitability of PCR for rapid caused by the West Nile virus, herpes simplex virus, and diagnosis, which allows for a more rapid initiation of anti- Japanese encephalitis virus has been reported for clinical viral therapy to improve patient outcomes. In the 65 patients application, based on the theory of increased viral clearance without characteristic manifestations of herpangina or HFMD, due to antibody-dependent neutralization and/or modulation viral isolation was positive in only 8 patients (12.3%), and for the inflammatory conditions (23,37,38). However, the viral RNA was detected by PCR in only 11 (16.9%). clinical efficacy of IVIg in enteroviral encephalitis remains Antibodies play a key role in the host defense against EVs. controversial (9). IVIg is prepared from pools of plasma samples obtained from As shown in Table 1, of the 18 virus-culture confirmed at least 1,000 healthy donors by the Cohn alcohol fraction- enteroviral encephalitis cases, the virus was most frequently ation method; thus, IVIg covers numerous arrays of antibodies obtained from stool and rectal swabs, but less from throat for variable viruses that comprise a broad range of immune swabs and CSF, and least from urine. No EV 71 infection antibodies directed against pathogens (13-16,22,23). Patients was identified in any of the specimens. In addition, EV 71 with agammaglobulinemia are at risk of developing severe VPI-IgM Rapid tests were negative in all of the patients. The and/or chronic enteroviral infections, and their condition may above tests might support the absence of EV 71 infections in be improved with administration of IVIg (24,25). In new- this study. However, physicians should be aware that RT-PCR borns, the absence of type-specific antibodies is a risk factor can detect EV71 at a higher rate in throat swabs, stools, and/ for the development of symptomatic enteroviral infections. or rectal swabs than in CSF specimens (39). Consequently, Since commercial IVIg products contain neutralizing anti- we strongly recommend collecting other clinical specimens bodies to commonly circulating enteroviral serotypes (12,24- (such as throat swabs, stool swabs, and rectal swabs) in addi- 27), IVIg has been used for prophylaxis during nursery out- tion to CSF and their simultaneous testing by RT-PCR and breaks (11-13,24). In addition to increased viral clearance virus culture to increase the detection rate for enteroviral due to antibody-dependent neutralization, the efficacy of encephalitis. high-dose IVIg has been demonstrated in a wide range of HEV B species are the most common cause of aseptic men- additional autoimmune diseases, including Guillain-Barré ingitis. They include all echoviruses, six serotypes of syndrome, myasthenia gravis, Kawasaki disease, systemic , coxsackievirus A9, and a number of newer , and systemic erythematosus (22,28-31). EVs (8,40). The predominant EV types vary from year to Although a complete definition of the mechanism of IVIg year, with echovirus 30, echovirus 13, and echovirus 18 being action is still lacking, extensive research suggests that IVIg the most frequent in Europe and the United States over the may achieve its therapeutic effects through multiple mechan- past few years (40). Figure 2 summarizes the distribution of isms of immunomodulation (22). For example, several pro- EV species in patients with culture-confirmed enteroviral posed mechanisms dependent on: (i) the antigen-binding encephalitis in our hospital from 1999 through 2006, and it (Fab) regions only (e.g., immunomodulation mediated by suggests that the main cause of viral encephalitis after the anti-idiotypic Fab within IVIg) (32); (ii) the Fc regions only 1998 EV 71 epidemic was HEV B species. Interestingly, we (e.g., competitive blockade of Fcγ receptors mediated by Fc have also found that serotypes varied during these years in within IVIg) (33); (iii) Fab and Fc regions (e.g., inhibition of Taiwan, as in Europe and the United States. Coxsackievirus autoantibody production by crosslinking the B-cell receptor B1, B2, B3, and B5 were found primarily in 1999. and Fcγ receptor IIB) (34); or (iv) IVIg contaminants (e.g., Coxsackievirus A9, B1, and B3 and echovirus 4, 9, 24, and immunosuppression mediated by soluble cytokine receptors 30 were found to co-circulate in 2000. Echovirus 30 and 11

22 caused local outbreaks in 2001 and 2003, respectively, which of enterovirus infection by automated RNA extraction and real-time is compatible with the report by Chen et al. of a disease out- fluorescence PCR. J. Clin. Virol., 25, 155-164. 7. Rotbart, H.A. (1995): Meningitis and encephalitis. p. 271-289. In H. A. break due to echovirus 30 with mosaic genome structure in Rotbart (ed.), Human Enterovirus Infections. ASM Press, Washington, Taiwan in 2001 (41). Outbreaks typically peak during the D.C. summer and early fall. The outbreak of echovirus 11 in 2003 8. Stanway, G. (2005): Family Picornaviridae. p. 757-758. In C.M. Fauquet, explains why the majority of patients with enteroviral M.A. Mayo, J. Maniloff, et al. (ed.), Virus Taxonomy. Eighth Report of the International Committee on Taxonomy of Viruses. Elsevier/Academic encephalitis in the present study were infected with HEV B Press, London. species instead of HEV A species, which is the main cause of 9. Jochem, M.D., Maria, T.E., Gerco, H.J., et al. (1997): Antibodies against HFMD and herpangina. Since encephalitis was diagnosed in enteroviruses in intravenous Ig preparations: great variation in titres all patients in the present study, it is not surprising to find that and poor correlation with the incidence of circulating serotypes. J. Med. HEV B was the main cause of this disease, even in group A Virol., 53, 273-276. 10. Melnick, J.L. (1996): Enteroviruses: , coxsackieviruses, patients with clinical manifestations of HFMD or herpangina. echoviruses, and newer enteroviruses. p. 655-712. In B.N. Fields, D.M. Epilepsy as a sequel of encephalitis was found in one group Knipe, P.M. Howley (ed.), Virology. Lippincott-Raven Press, Philadel- C patient, whose evaluation by RT-PCR (establishing echo- phia. virus 11 as the cause) was not undertaken until late in the 11. Nagington, J. (1982): Echovirus 11 infection and prophylactic anti- serum [letter]. Lancet, 1, 446. study. The epilepsy in this case might have been prevented 12. Carolane, D.J., Long, A.M., McKeever, P.A., et al. (1985): Prevention by earlier administration of IVIg, and conducting RT-PCR of spread of echovirus 6 in a special care baby unit. Arch. Dis. Child., testing soon after admission might have resulted in earlier 60, 674-676. treatment. In Taiwan, IVIg is the treatment indicated for pa- 13. Nagington, J., Gandy, G., Walker, J., et al. (1983): Use of normal immuno- tients with severe enteroviral infections including encephalitis globulin in an echovirus 11 outbreak in a special-care baby unit. Lancet, 2, 443-446. (4). This strategy is controversial (9). The present results, 14. Black, S. (1983): Treatment of overwhelming neonatal ECHO 5 virus however, support the use of this strategy, and suggest that infection with intravenous gamma globulin (abstract no. 314). p. 140. IVIg teamed with rapid diagnosis using RT-PCR may shorten In S. Black (ed.), Program and Abstracts of the 23rd Interscience hospital stays and improve outcomes. Conference on Antimicrobial Agents and Chemotherapy (Las Vegas). ASM Press, Washington, D.C. In the present study, IVIg was administered as indicated 15. Johnston, J.M. and Overall, J.C., Jr. (1989): Intravenous immuno- for enteroviral infection by the CDC of Taiwan (4). Addi- globulin in disseminated neonatal echovirus 11 infection. Pediatr. tionally, the consensus for IVIg therapy of other viral encepha- Infect. Dis. J., 8, 638-641. litides remains unclear; therefore, patients who are culture- 16. Valduss, D., Murray, D.L., Karna, P., et al. (1993): Use of intravenous positive for viruses other than EVs are not treated with IVIg. immunoglobulin in twin neonates with disseminated coxsackie B1 infec- tion. Clin. Pediatr. (Phila.), 32, 561-563. However, patients infected with influenza A or B virus, 17. Romero, J.R. (1999): Reverse-transcription polymerase chain reaction herpes simplex virus, and cytomegalovirus are treated with detection of the enteroviruses. Arch. Pathol. Lab. Med., 123, 1161-1169. oseltamivir, acyclovir, and gancyclovir, respectively; all of 18. Rotbart, H.A. (1997): Reproducibility of AMPLICOR enterovirus PCR these patients achieve good outcomes without any sequelae. test results. J. Clin. Microbiol., 35, 3301-3302. 19. Pozo, F., Casas, I., Tenorio, A., et al. (1998): Evaluation of a commer- Specific antiviral drugs for EVs are not currently available, cially available reverse transcription-PCR assay for a diagnosis of which emphasizes the importance of effective adjuvant treat- enteroviral infection in archival and prospectively collected cerebrospi- ment for severe enteroviral infections. In addition, IVIg, nal fluid specimens. J. Clin. Microbiol., 36, 1741-1745. steroid pulse therapy has also been reported to treat acute viral 20. Lina, B., Pozzetto, B., Andreoletti, L., et al. (1996): Multicenter evalua- encephalitis (42,43). However, the effect remains uncertain tion of a commercially available PCR assay for diagnosing enterovirus infection in a panel of cerebrospinal fluid specimens. J. Clin. Microbiol., (44,45) and is not currently recommended in the guidelines 34, 3002-3006. for treatment of severe enteroviral infection in Taiwan, and 21. Nitsche, A., Steuer, N., Schmidt, C.A., et al. (1999): Different real-time thus was not considered in the study design. Though the PCR formats compared for the quantitative detection of cytomegalovirus present study supports the strategy of using IVIg teamed with DNA. Clin. Chem., 45, 1932-1937. 22. Jin, F. and Balthasar, J.P. (2005): Mechanisms of intravenous immuno- rapid diagnosis by RT-PCR, double-blind, randomized, globulin action in immune . Hum. Immunol., placebo-controlled trials using larger patient populations will 66, 403-410. be needed to confirm the efficacy of this strategy for treating 23. Caramello, P., Canta, F., Balbiano, R., et al. (2006): Role of intravenous enteroviral encephalitis at an earlier stage. immunoglobulin administration in Japanese encephalitis. Clin. Infect. Dis., 43, 1620-1621. 24. Abzug, M.J., Keyserling, H.L., Lee, M.L., et al. (1995): Neonatal ACKNOWLEDGMENTS enterovirus infection: virology, serology, and effects on intravenous This work was supported by a grant from the Veterans General Hospital- immune globulin. Clin. Infect. Dis., 20, 1201-1206. Kaohsiung, Taiwan (VGHKS93-74). 25. McKinney, R.E., Jr., Katz, S.L. and Wilfert, C.M. (1987): Chronic enteroviral meningoencephalitis in agammaglobulinemic patients. Rev. Infect. Dis., 9, 334-356. REFERENCES 26. Dagan, R., Prather, S.L., Powell, K.R., et al. (1983): Neutralizing anti- 1. Ho, M., Chen, E.R., Hsu, K.H., et al. (1999): An epidemic of enterovirus bodies to non- enteroviruses in human immune serum globulin. 71 infection in Taiwan. N. Engl. J. Med., 341, 929-935. Pediatr. Infect. Dis., 2, 454-456. 2. Lin, T.Y., Twu, S.J., Ho, M.S., et al. (2003): Enterovirus 71 outbreaks, 27. Keyseling, H.L. and Torfason, E.G. (1986): Comparison of intravenous Taiwan: occurrence and recognition. Emerg. Infect. Dis., 9, 291-293. gamma globulin preparations in neutralization assays against entero- 3. Wang, J.R., Tsai, H.P., Chen, P.F., et al. (2000): An outbreak of viruses (abstract no. 328). p. 156. In Program and Abstracts of the 26th enterovirus 71 infection in Taiwan, 1998. II. Laboratory diagnosis and Interscience Conference on Antimicrobial Agents and Chemotherapy genetic analysis. J. Clin. Virol., 17, 91-99. (New Orleans). ASM Press, Washington, D.C. 4. Center for Disease Control, Taiwan (2006): Guidelines for treatment of 28. Arsura, E. (1989): Experience with intravenous immunoglobulin in severe enteroviral infection. p. 11. Clinical management and indication myasthenia gravis. Clin. Immunol. Immunopathol., 53, S170-179. of IVIg usage for severe enterovirus infection (in Chinese). 29. Jordan, S.C. (1989): Intravenous gamma-globulin therapy in systemic 5. Rotbart, H.A. and Romero, J.R. (1995): Laboratory diagnosis of enteroviral lupus erythematosus and immune complex disease. Clin. Immunol. infections. p. 401-410. In H.A. Rotbart (ed.), Human Enterovirus In- Immunopathol., 53, S164-169. fections. ASM Press, Washington, D.C. 30. Jayne, D.R., Davies, M.J., Fox, C.J., et al. (1991): Treatment of systemic 6. Holger, F.R., Alexandra, C., Gerhard, M., et al. (2002): Rapid detection vasculitis with pooled intravenous immunoglobulin. Lancet, 337, 1137-

23 1139. possible infection via trigeminal nerve. Clin. Neurol., 45, 293-297 (in 31. Newburger, J.W., Takahashi, M., Burns, J.C., et al. (1986): The treatment Japanese). of Kawasaki syndrome with intravenous gamma globulin. N. Engl. J. 39. Tsao, L.Y., Lin, C.Y., Yu, Y.Y., et al. (2006): Microchip, reverse Med., 315, 341-347. transcription-polymerase chain reaction and culture methods to detect 32. Berchtold, P., Dale, G.L., Tani, P., et al. (1989): Inhibition of autoanti- enterovirus infection in pediatric patients. Pediatr. Int., 48, 5-10. body binding to platelet glycoprotein IIb/IIIa by anti-idiotypic antibodies 40. Thoelen, I., Moes, E., Lemey, P., et al. (2004): Analysis of the serotype in intravenous gammaglobulin. Blood, 74, 2414-2417. and genotype correlation of VP1 and the 5´ noncoding region in an 33. Bussel, J.B. (2002): Another interaction of the FcR system with IVIG. epidemiological survey of the human enterovirus B species. J. Clin. Thromb. Haemost., 88, 890-891. Microbiol., 42, 963-971. 34. Ballow, M. (1997): Mechanisms of action of intravenous immune serum 41. Chen, G.W., Huang, J.H., Lo, Y.L., et al. (2007): Mosaic genome struc- globulin in autoimmune and inflammatory diseases. J. Allergy Clin. ture of echovirus type 30 that circulated in Taiwan in 2001. Arch. Virol., Immunol., 100, 151-157. [Epub ahead of print]. 35. Sewell, W.A. and Jolles, S. (2002): Immunomodulatory action of intra- 42. Nakano, A., Yamasaki, R., Miyazaki, S., et al. (2003): Beneficial effect venous immunoglobulin. Immunology, 107, 387-393. of steroid pulse therapy on acute viral encephalitis. Eur. Neurol., 50, 36. Wang, S.M., Lei, H.Y., Huang, M.C., et al. (2006): Modulation of 225-229. cytokine production by intravenous immunoglobulin in patients with 43. Koyama, S., Morita, K., Yamaguchi, S., et al. (2000): An adult case of enterovirus 71-associated brainstem encephalitis. J. Clin. Virol., 37, 47- brainstem encephalitis. Intern. Med., 39, 499-502. 52. 44. Johnson, R.T., Intralawan, P. and Puapanwatton, S. (1986): Japanese 37. Haley, M., Retter, A.S., Fowler, D., et al. (2003): The role for intra- encephalitis: identification of inflammatory cells in cerebrospinal fluid. venous immunoglobulin in the treatment of West Nile virus encephalitis. Ann. Neurol., 20, 691-695. Clin. Infect. Dis., 37, 88-90. 45. Matsui, M. (1999): Infection in the central nervous system and cortico- 38. Yoshidome, Y., Hayashi, S. and Maruyama, Y. (2005): A case of brainstem steroid therapy. Clin. Neurol., 39, 26-28 (in Japanese). encephalitis caused by herpes simplex virus type 1 with

24