Journal of Perinatology (2009) 29, 290–296 r 2009 Nature Publishing Group All rights reserved. 0743-8346/09 $32 www.nature.com/jp ORIGINAL ARTICLE Persistence of DNA in cerebrospinal fluid of neonates with encephalitis

A Mej´ıas1, R Bustos2, MI Ardura1, C Ram´ırez1 and PJ Sa´nchez1 1Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA and 2Department of Pediatrics, University of Concepcio´n, Chile

outcome in the majority of survivors.4 Traditional risk factors that Objective: The significance of detecting herpes simplex virus (HSV) DNA contribute to increased morbidity in these infants include the in the cerebrospinal fluid (CSF) of infants with HSV encephalitis after presence of seizures, duration of illness (less than 2 or greater À1 À1 receipt of prolonged therapy with high-dose (60 mg kg day ) acyclovir than 8 days) before the initiation of therapy and infection with is unknown. We report the clinical and characteristics, HSV-II virus.5,6 neuroimaging studies and outcomes of four neonates with HSV encephalitis Polymerase chain reaction (PCR) for HSV DNA performed on who had persistence of CSF HSV DNA, by polymerase chain reaction (PCR) cerebrospinal fluid (CSF) is the best available method for diagnosis after 15 to 21 days of high-dose acyclovir therapy. of HSV CNS infection.7,8 The occurrence of a positive CSF HSV Study Design: Retrospective chart review. DNA PCR after receipt of prolonged therapy with high-dose (60 mg kgÀ1 per day) acyclovir has not been reported in detail and Results: All four infants had abnormal neuroimaging studies and its significance remains unknown. For this reason, we describe four subsequently experienced severe developmental delay or death. neonates with HSV encephalitis, three of whom were born in Texas Conclusion: A persistently positive CSF HSV PCR in neonates may be and one in Chile, who had persistence of CSF HSV DNA by PCR another risk factor for worse neurodevelopmental outcome. Prospective after 14 to 21 days of high-dose acyclovir therapy. This report also studies are needed to document how often HSV DNA persists in CSF, describes the adverse outcomes that these infants experienced, and elucidate whether it represents an initially high CSF , ongoing it highlights the need for prospective studies to document how often or viral resistance, and determine its possible association HSV DNA persists in CSF of infected infants and understand its with neurodevelopmental impairment. prognostic significance. Journal of Perinatology (2009) 29, 290–296; doi:10.1038/jp.2008.235; published online 5 February 2009 Patients Keywords: HSV; PCR; encephalitis; acyclovir; neonate Case 1 A 2860-gram female infant was delivered vaginally at 36 weeks of gestation to a 29-year-old gravida4, para3003 Caucasian woman. Prenatal serologic tests revealed an absence of antibodies to the Introduction human immunodeficiency virus, a nonreactive rapid plasma B Neonatal herpes simplex virus (HSV) infection occurs in 1in reagin test, negative hepatitis B surface antigen and a negative 1250 to 20 000 neonates per year in the United States1,2 with 3 screening culture for group B streptococcus. There was no history approximately 30% having encephalitis. Although acyclovir of genital HSV infection. Rupture of fetal membranes occurred therapy has reduced significantly the mortality in infants with 24 h before delivery, and a scalp electrode was used for fetal heart herpes encephalitis, central nervous system (CNS) infection with rate monitoring. HSV continues to be associated with abnormal neurodevelopmental The infant was well until 15 days of age when she developed Correspondence: Dr Asuncio´n Mejı´as, Department of Pediatrics, Division of Pediatric focal seizures. She was hospitalized at 18 days of age at Children’s Infectious Diseases, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Medical Center Dallas (TX, USA) for persistent seizures and apneic Dallas, TX 75390-9063, USA. episodes. The infant was afebrile and physical examination was E-mail: [email protected] significant for a small vesicle on the left inguinal area and This work was presented in part at the Pediatric Academic Societies Meeting, Toronto, hypertonicity. There was no hepatosplenomegaly or petechiae, and Canada from May 5 to 7, 2007. Received 14 July 2008; revised 8 December 2008; accepted 12 December 2008; published online liver function tests, bilirubin concentration and complete blood 5 February 2009 cell count were normal (Table 1). HSV-2 DNA PCR (Children’s CSF HSV persistence in neonatal encephalitis A Mejı´as et al 291

Table 1 . Clinical and laboratory characteristics of four neonates who had encephalitis due to herpes simplex virus (HSV), and the cerebrospinal fluid HSV polymerase chain reaction was persistently positive

Case 1 Case 2 Case 3 Case 4

Classification of HSV disease CNS CNS CNS CNS HSV type HSV-2 ND HSV-2 HSV-2 Age at onset of illness 15 days 28 days 2 hours 23 days Liver dysfunction No No No No Thrombocytopenia No No No No Seizures Yes Yes Yes Yes Duration of illness before initiation of acyclovir therapy (days) 3 10 <1 5 Age at onset of HD acyclovir 22 daysa 38 days 18 hours 28 days Hospital day of initiation of HD acyclovir 4 1 1 4 Duration of HD acyclovir therapy (days) 28 30 28 48 Neutropenia on acyclovir therapy No No No No

Abbreviations: CNS, central nervous system; HD, high dose; ND, not done. aInfant received acyclovir at 40 mg kgÀ1 per day for 4 days before high-dose acyclovir was started at 22 days of age.

Table 2 Day of high-dose (60 mg kgÀ1 per day) acyclovir therapy and results of cerebrospinal fluid (CSF) studies in the four neonates with herpes simplex virus (HSV) encephalitis

Case 1 Case 2 Case 3 Case 4

Day of high-dose acyclovir treatment 1a 18b 28b 1a 20 1a 515261a 21 38 CSF HSV polymerase chain reaction + + À ++NS+ +À ++À Cerebrospinal fluid indices White blood cells per mm3 Total ( Â 106 per mm3) 83 88 38 38 200 82 53 32 26 140 38 44 Neutrophils (%) 8 1 0 3 2 76 4 6 6 56 20 ND Lymphoctyes (%) 28 91 80 80 98 10 62 67 79 44 80 ND Monocytes (%) 64 7 20 17 0 14 27 9 24 0 0 ND Red blood cells per mm3 267 703 25 51 10 600 <1 10 690 4 640 6 500 2 5 2 Protein (mg per 100 ml) 235 613 220 194 628 113 NS 480 217 45 33 231 Glucose (mg per 100 ml) 38 30 38 31 30 61 NS 32 43 42 34 68

Abbreviations: ND, not done; NS, not significant. aDay 1 reflects the results of the lumbar puncture before acyclovir therapy was initiated. bDays of high-dose acyclovir after infant had received 4 days of acyclovir at 40 mg kgÀ1 per day.

Medical Center Dallas) performed on the initial CSF specimen was repeated 28 days after initiation of high-dose acyclovir. CSF and blood was positive (Table 2). HSV-2 PCR was negative, and high-dose acyclovir was discontinued Acyclovir was provided at a dose of 40 mg kgÀ1 per day at 54 days of age and after a total of 32 days. Acyclovir suppressive intravenously for 4 days and subsequently increased to 60 mg kgÀ1 therapy was not provided. per day. Viral cultures of the throat and vesicle did not yield HSV. The patient’s serum white blood cell count and creatinine Bacterial cultures of blood and CSF were sterile. concentration remained normal during acyclovir therapy. MRI of the Computed tomography (CT) and magnetic resonance imaging brain performed on day 22 of acyclovir (44 days of age) showed (MRI) of the brain at presentation showed extensive cytotoxic megacystic encephalomalacia with laminar necrosis, gliosis and edema involving both cerebral hemispheres. Electroencephalogram cortical atrophy with parieto-occipital subdural effusions (Figure 1a). performed on hospital admission showed extensive multifocal On follow-up at 5, 11, 16 and 24 months of age, she had severe sharp-wave activity with absence of normal background. developmental delay with hypertonicity and microcephaly. She has Ophthalmologic and hearing assessments were normal. not experienced any skin or CNS recurrences. At 40 days of age, after 18 days of high-dose acyclovir therapy, a second CSF examination revealed the persistence of HSV DNA by Case 2 PCR (Table 2). The infant was clinically stable without further A 2930-gram, 40-week female infant was delivered vaginally to a seizure activity. Acyclovir was continued and a lumbar puncture 23-year-old gravida4, para3003 African-American woman. There was

Journal of Perinatology CSF HSV persistence in neonatal encephalitis A Mejı´as et al 292

Case 1 Case 2

Case 3 Case 4

Figure 1 Neuroimaging studies of the brain in four neonates diagnosed with herpes simplex virus encephalitis. (a and b) Magnetic resonance imaging (MRI) of the brain performed on week 3 (Case 1) and 1 (Case 2) of high-dose acyclovir, respectively, showed megacystic encephalomalacia with laminar necrosis and gliosis, and cortical atrophy with parieto-occipital subdural effusions. (c) MRI of the brain performed on day 5 of high-dose acyclovir showed diffuse supratentorial edema with large regions of restricted diffusion and a possible thrombus in the right transverse sinus. (d) Computed tomography of the brain performed without contrast on day 48 of high-dose acyclovir therapy showed severe bilateral encephalomalacia. no history of genital HSV infection but she was treated for and CSF, as well as viral cultures of throat, rectum and buffy coat gonococcal and chlamydial infections in the first trimester of were sterile. The infant received intravenous ampicillin, pregnancy. She lacked antibodies to human immunodeficiency cefotaxime, acyclovir (60 mg kgÀ1 per day) and oral nystatin. On virus and had nonreactive and hepatitis B admission, CT scan without contrast showed subdural effusions surface antigen tests. Rupture of fetal membranes occurred at of both frontal areas that extended over the left frontal parietal delivery, and it is not known whether a fetal scalp electrode convexity, small subdural infratentorial hemorrhages, and diffusely was used. abnormal low density of white matter and basal ganglia. The infant was well until 28 days of age when she developed Electroencephalogram did not reveal epileptiform activity. hypersommnolence, decreased oral intake, diarrhea and focal Ophthalmologic examination showed bilateral chorioretinitis. seizures. Because of persistent seizures and lethargy, she was On the 7 day of hospitalization, brain MRI showed diffuse cystic referred to Children’s Medical Center Dallas where she was encephalomalacia of both cerebral hemispheres and bilateral hospitalized at 38 days of age. On admission the infant was subdural hygromas (Figure 1b). At 58 days of age, on the 20th day afebrile, and physical examination revealed horizontal nystagmus, of acyclovir therapy, a second CSF examination revealed worse generalized hypertonia as well as oral candidiasis, but no CSF indices and CSF HSV PCR remained positive (Table 2). hepatosplenomegaly, petechiae or skin vesicles. CSF HSV PCR The infant’s hospital course was marked by progressive (ARUP , Salt Lake City, UT, USA) was positive but the neurological deterioration with lethargy and minimal response to HSV type was not performed (Table 1). Bacterial cultures of blood tactile stimulation, as well as intermittent fever and inappropriate

Journal of Perinatology CSF HSV persistence in neonatal encephalitis A Mejı´as et al 293 production of antidiuretic hormone resulting in hyponatremia. On A cranial ultrasound performed on day 12 of acyclovir treatment the third week of hospitalization, she developed opisthotonus and showed increased echogenicity of the gray and white matter, basal decerebrate posturing, and required nasogastric tube feedings. At ganglia and thalami, with cystic changes in the white matter of this time, a head CT scan showed extensive cortical hemorrhage both cerebral hemispheres compatible with encephalomalacia. On with near dissolution of the white matter. At 68 days of age, the 25th day of acyclovir therapy at 25 days of age a third CSF acyclovir was discontinued after 30 days of therapy without evaluation was performed, and when the HSV PCR result was confirmation of a negative CSF HSV PCR test, as multiple attempts reported as negative acyclovir was discontinued after 28 days. The at performing a lumbar puncture were unsuccessful. The patient’s infant developed fulminant necrotizing enterocolitis (Bell Stage white blood cell count and creatinine remained normal during IIIA) and died 3 days later. The parents declined a postmortem treatment with acyclovir. Suppressive therapy with oral acyclovir examination. was not prescribed. Follow-up evaluations at the ages of 2 and 10 months, 2, 3 and Case 4 4 years of age revealed microcephaly, spastic quadriplegia and A 3710-gram, 40-week male infant was born vaginally to a severe developmental delay. Brain MRI at 4 years of age showed 28-year-old gravida2,para0010 Hispanic mother following 8 h of cystic encephalomalacia. rupture of membranes. Prenatal serologic testing was negative for human immunodeficiency virus antibodies, as well as rapid plasma Case 3 reagin and hepatitis B surface antigen tests. The mother had no A 2875-gram, 39-week male infant was born vaginally to a history of genital HSV infection, and a fetal scalp electrode was not 21-year-old gravida2, para1001 Caucasian woman following 1½ h of used intrapartum. The infant was well up to 24 days of age when rupture of membranes. Prenatal testing was negative for human he was hospitalized at Hospital Guillermo Grant Benavente in immunodeficiency virus antibodies, rapid plasma reagin and Concepcion, Chile with 1-day history of fever, irritability, poor hepatitis B surface antigen tests, and group B streptococcus oral intake and generalized tonic-clonic seizures. On physical colonization. She had no history of genital HSV infection. examination there were no hepatosplenomegaly or skin lesions Meconium-stained amniotic fluid was noted intrapartum and a (Table 1). CSF examination only revealed pleocytosis. CT scan of the scalp electrode was used to monitor the fetal heart rate. The infant brain was normal, and the infant received ampicillin and amikacin. had Apgar scores of 8 and 9 at 1 and 5 min, respectively. Within 2 h Bacterial cultures of blood and CSF were sterile, but on the fourth of birth, he developed apnea and focal seizures, and he required hospital day he experienced intractable seizures and a second lumbar mechanical ventilation in the neonatal intensive care unit at puncture was performed that again showed pleocytosis (Table 2); Parkland Memorial Hospital (Dallas, TX, USA) (Table 1). The CSF HSV-II PCR (LightCycler HSV1/2 Detection Kit, Roche infant was afebrile and the physical examination was significant Diagnostics, performed at the Molecular Biology Laboratory, for nasal flaring, poor peripheral perfusion, eye deviation and lip Concepcion University, Chile) was positive. A second CT scan of the smacking. There were no vesicular skin lesions. Chest radiograph brain now showed diffuse cerebral edema, and intravenous acyclovir À1 was normal. There was insufficient quantity of CSF for viral culture at a dose of 60 mg kg per day was initiated. At 42 days of age, an and HSV PCR. ophthalmologic examination performed on the 14th day of treatment The infant received initially ampicillin and gentamicin, but revealed bilateral chorioretinitis and vasculitis with peripheral when the CSF indices were assessed to be abnormal at 18 h of age exudative retinopathy. At 49 days of age, on the 21st day of high-dose (Table 2), treatment with cefotaxime and acyclovir (60 mg kgÀ1 acyclovir treatment, a third CSF evaluation revealed a positive HSV per day) was provided. A repeat CSF evaluation on day 4 of PCR (Table 2). At this time, his neurological examination revealed acyclovir therapy was positive for HSV-II DNA by PCR (Mayo severe hypotonia with a Glasgow coma scale of 10 to 11. Medical Labs, Rochester, MN, USA). Viral cultures of CSF, throat, At 66 days of age, on day 38 of acyclovir therapy, CSF HSV PCR rectum and buffy coat were sterile. was negative and acyclovir was discontinued after a total of MRI of the brain performed on day 5 of acyclovir therapy 48 days. At this time, CT scan of the brain showed severe bilateral showed diffuse supratentorial edema with large regions of restricted encephalomalacia (Figure 1d). The infant did not receive oral diffusion and a possible thrombus in the right transverse sinus suppressive acyclovir therapy and there were no known CNS (Figure 1c). Electroencephalogram showed continuous seizure recurrences. However, on follow-up at 12 months of age the patient activity. Ophthalmologic and hearing evaluations were normal. has severe developmental delay and epilepsy. On the 14th day of acyclovir therapy at 14 days of age, the CSF HSV-II PCR remained positive. The infant experienced more clinical seizures as well as feeding intolerance, and Discussion phosphenytoin was added to the initial anticonvulsant therapy Since 1941, when Smith and Reames9 first isolated HSV from with phenobarbital. the brain of a neonate with encephalitis, HSV has been a leading

Journal of Perinatology CSF HSV persistence in neonatal encephalitis A Mejı´as et al 294 cause of viral CNS infections in neonates. In recent years, the HSV disease treated for 21 days had a significantly higher survival diagnosis of HSV encephalitis has been greatly improved and rate than similar infants from the previous Collaborative Antiviral facilitated by the development of PCR that detects HSV DNA in CSF. Study Group trial that utilized acyclovir at a dose of 30 mg kgÀ1 Its sensitivity and specificity range from 75 to 100% and 71 to per day for 10 days. In addition, recipients of high-dose acyclovir 100%, respectively10–12 but its validity is complicated by were seven times as likely to be developmentally normal at 12 interlaboratory variability. months of age as patients who received the lower dose of acyclovir. Lakeman and Whitley10 of the NIAID’s Collaborative Antiviral On the basis of these findings, longer duration of therapy and a Study Group evaluated the use of PCR in 101 adult patients with higher dose of acyclovir are recommended for treatment of biopsy-proven herpes encephalitis diagnosed between 1974 and neonatal HSV infection.20 1987.13–15 These patients had participated in several randomized, The occurrence of a positive CSF HSV PCR after 2 to 3 weeks of placebo-controlled trials of iodoxouridine, vidarabine or acyclovir, high-dose acyclovir therapy is known but it has not been well the latter provided at a dose of 30 mg kgÀ1 per day. During the first documented. It is possible that the persistently positive PCR reflects week of antiviral therapy, 49 (98%) of 50 CSF specimens from 39 an initially high CSF viral load that is reflective of a greater degree patients were HSV DNA-positive by PCR. By the second week of of CNS injury and results in a longer time for achievement of an therapy, HSV DNA was detected by PCR in 47% (8/17) of available undetectable concentration.21 Domingues et al.22 found that CSF specimens. After 2 weeks of antiviral treatment, CSF samples among 16 patients with HSV-1 encephalitis who had a mean age of from 4 (22%) of 18 subjects remained positive by PCR, 3 patients 37±21 years, greater than 100 copies of HSV DNA per ml in CSF had received vidarabine and 1 was treated with acyclovir. A positive was associated with decreased level of consciousness, presence of CSF HSV PCR was not associated with any abnormal clinical or lesions detected by CT scan of brain and poor neurological laboratory findings such as level of consciousness, seizures, outcomes. Similar studies that assess the contribution of HSV viral dysphasia, CSF indices, electroencephalogram abnormalities or load to neurodevelopmental outcome or antiviral response are Glasgow coma scale. No assessment of long-term abnormalities needed in neonates. Because the HSV DNA PCR that was performed was performed. in our patients was not quantitative, we were unable to correlate The Collaborative Antiviral Study Group also performed a viral load with abnormal neurodevelopment. In addition, the lack prospective, randomized trial of intravenous acyclovir versus of CSF HSV cultures in our patients did not allow us to evaluate vidaribine, both administered at a dose of 30 mg kgÀ1 per day for whether the persistently positive HSV PCR may have been due to 10 days, in 202 neonates with HSV infection.16 A retrospective the presence of live virus in CSF. testing of stored CSF specimens obtained from 30 of these study Persistence of HSV DNA in CSF may be secondary to acyclovir subjects after completion of antiviral therapy found that 14 (74%) resistance, which occurs in 5% of immunocompromised patients of 18 neonates with HSV encephalitis, as well as all 5 (100%) who and 0.1 to 0.6% of immunocompetent individuals.23 Acyclovir had disseminated disease that involved the CNS, but none of the 7 resistance has been reported in three cases of neonatal HSV-2 who had skin, eye or mouth disease, had a positive CSF HSV infection: (1) full-term infant with infection of the larynx who PCR.11,16 The majority of these CSF specimens were obtained recovered despite positive HSV cultures after 11 days of foscarnet within 10 days after antiviral therapy was discontinued, and HSV therapy,24 (2) preterm infant with congenital disseminated disease was not isolated from the 15 infants who had CSF viral cultures in which acyclovir resistance developed during the first 7 days of performed. In addition, of the 30 study subjects, 13 (87%) infants treatment (30 mg kgÀ1 per day) and who died despite foscarnet treated with vidaribine had HSV DNA detected in post-therapy CSF therapy,25 and (3) preterm infant who developed disseminated compared with 6 (40%) of the 15 who had received acyclovir disease 8 days after receiving 21 days of high-dose acyclovir for (P ¼ 0.008). The detection of HSV DNA from post-therapy CSF was neurocutanous disease. This latter infant also had a positive CSF associated with moderate-to-severe neurological impairment or HSV PCR at 48 h after completion of the initial 21 days of death in 95% of PCR-positive infants. Similarly, Kimura et al17 acyclovir.26 In addition, acyclovir resistance was reported in an reported the persistence of HSV DNA in CSF of three of five neonates infant who had a cutaneous HSV-2 recurrence 36 h after cessation with HSV disease that involved the CNS after receipt of 7 to 14 days of 6 months of acyclovir suppression.27 These cases highlight the of acyclovir at a dose of 30 mg kgÀ1 per day. These findings have importance of obtaining herpes culture or performing genotypic resulted in the recommendation that patients with HSV CNS characterization of PCR products for acyclovir resistance testing in infection have a CSF HSV PCR performed before the end of infants who do not respond appropriately to acyclovir therapy, have antiviral therapy, and that treatment be continued until it is frequent recurrences while on acyclovir suppressive therapy or were negative.18 born to mothers who received prolonged acyclovir suppression Subsequently, Kimberlin et al.19 found that treatment of during pregnancy. Foscarnet therapy is recommended for disease neonatal HSV infection with a higher dose of acyclovir (60 mg kgÀ1 caused by an acyclovir-resistant isolate.28 Unfortunately, per day) improved outcome.19 Infants with CNS or disseminated determination of acyclovir resistance was not performed in our

Journal of Perinatology CSF HSV persistence in neonatal encephalitis A Mejı´as et al 295 patients, although it is unlikely as three of the four patients 8 Barbi M, Binda S, Primache V, Tettamanti A, Negri C, Brambilla C. Use of Guthrie eventually had a negative HSV CSF PCR without the addition of cards for the early diagnosis of neonatal herpes simplex virus disease. Pediatr Infect foscarnet. Dis J 1998; 17(3): 251–252. A positive HSV CSF PCR also may represent ongoing viral 9 Smith MG LE, Reames HR. of the virus of herpes simplex and the demostration of intranuclear inclusions in a case of acute encephalitis. Am J Pathol replication that is not controlled satisfactorily by a neonate’s 1941; 17: 55–68. 29 immature or defective immune system. Recently, Casrouge et al. 10 Lakeman FD, Whitley RJ. Diagnosis of herpes simplex encephalitis: application of reported that children who lack functional UNC-93B, an polymerase chain reaction to cerebrospinal fluid from brain-biopsied patients and endoplasmic reticulum protein required for toll-like receptor correlation with disease. National Institute of Allergy and Infectious Diseases signaling, or have mutations in toll-like receptor-3 are predisposed Collaborative Antiviral Study Group. J Infect Dis 1995; 171(4): 857–863. 11 Kimberlin DW, Lakeman FD, Arvin AM, Prober CG, Corey L, Powell DA et al. to develop HSV encephalitis through impaired type-I interferon Application of the polymerase chain reaction to the diagnosis and management 30 responses. Our patients were not tested for such defects, although of neonatal herpes simplex virus disease. National Institute of Allergy and this should need to be considered in the future. Infectious Diseases Collaborative Antiviral Study Group. 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