Viral Central Nervous System Infections in Infants <90 Days Old: A Paediatric Investigators’ Collaborative Network on Infections in Canada (PICNIC) Study


 Background The relative contribution of viruses to central nervous system (CNS) infections in young infants is not clear. For viral CNS infections, there are limited data on features that suggest HSV etiology or that are predictors of unfavorable outcome. Methods In this cross-sectional study, the Pediatric Investigators Collaborative Network on Infections in Canada identified infants <90 days of age with CNS infection proven to be due to viruses. Results Of 174 proven CNS infections, viral causes accounted for 111 (64%) including enterovirus (EV) (N=103; 93%), HSV (N=7; 6%) and human parechovirus (HPeV) (N=1; 1%). All HSV cases and 45 (43%) non-HSV cases presented before 21 days of age. HSV cases were more likely to require ICU admission (p=0.010), present with seizures (p=0.031) and have extra-CNS disease (p<0.001). Three HSV cases (43%) did not have seizures while 4 (57%) HSV and 33 (32%) non-HSV cases lacked cerebrospinal fluid (CSF) pleocytosis. Conclusions Viruses account for about half of proven CNS infections in the first 90 days of life. Empiric therapy for HSV should be considered in suspected CNS infections in the first 21 days even in the absence of CSF pleocytosis unless CSF parameters are suggestive of bacterial meningitis.


Abstract Background
The relative contribution of viruses to central nervous system (CNS) infections in young infants is not clear. For viral CNS infections, there are limited data on features that suggest HSV etiology or that are predictors of unfavorable outcome.

Methods
In this cross-sectional study, the Pediatric Investigators Collaborative Network on Infections in Canada identi ed infants <90 days of age with CNS infection proven to be due to viruses.

Conclusions
Viruses account for about half of proven CNS infections in the rst 90 days of life. Empiric therapy for HSV should be considered in suspected CNS infections in the rst 21 days even in the absence of CSF pleocytosis unless CSF parameters are suggestive of bacterial meningitis.

Background
The prevention of bacterial meningitis by conjugate vaccines has resulted in viruses accounting for an increasing proportion of central nervous system (CNS) disease in childhood. 1 The improvement in viral diagnostics has made this trend more apparent. Previous studies of viral CNS disease were limited by small sample size, included cases where the etiology was not proven or did not focus on infants. 2−3 The most common viruses associated with CNS infections are enteroviruses (EV), which compared to bacterial CNS infections are less likely to lead to adverse neurologic outcome. By contrast, herpes simplex virus (HSV) CNS infections result in signi cant morbidity and mortality, especially if acyclovir is delayed. 4 It is therefore vital that clinicians know what clinical and laboratory features should lead them to start empiric acyclovir. This was a cross-sectional analysis to identify infants less than 90 days of age with proven CNS infections. We sought to a) determine the relative contribution of HSV and EV to microbiologicallycon rmed CNS infections, b) provide a comparative analysis of the epidemiology and outcome of HSV and non-HSV viral CNS infection, c) describe factors associated with HSV aetiology and d) identify factors associated with unfavorable outcome.

Study Population and Design:
Seven paediatric academic centres within the Paediatric Investigators Collaborative Network on Infections in Canada (PICNIC) retrospectively enrolled hospitalized infants <90 days of age with microbiologically-con rmed CNS infection January 1, 2013 through December 31, 2014. Cases were identi ed using appropriate codes from the International Statistical Classi cation of Diseases and Related Health Problems, Tenth Revision, Canada diagnostic codes for target discharge (Appendix A) and charts were then reviewed. A previous publication described cases of bacterial CNS infection as proven if bacteria was detected from cerebrospinal uid (CSF) or brain abscess by means of culture or PCR or probable if CSF pleocytosis was present, along with bacterial growth from another sterile site. 5 For the purposes of this study, we included all proven cases of viral CNS infection based on the identi cation of a virus in the CSF using viral culture or polymerase chain reaction (PCR) during life or in the brain tissue using PCR at autopsy. All study centres offered routine PCR testing for HSV and EV while only two centres offered HPeV testing during the study period. Cases with coinfection were excluded unless the investigator deemed that a virus was the main pathogen. There were no other exclusion criteria. Descriptive analysis was conducted. Chi-square or Fisher's exact test was used to compare categorical variables and non-parametric tests were used to compare continuous variables (Mann-Whitney U test). Exploratory analysis was conducted using univariate analyses and where sample size allowed, multivariate analyses to identify clinical, laboratory or outcome differences between HSV and non-HSV viral cases were conducted using factors identi ed as signi cant in univariate analysis. Additionally, we used univariate analysis to explore potential factors associated with an unfavorable outcome overall. Epiinfo version 7 was used for statistical analysis.

Relative contribution of viruses to microbiologicallycon rmed CNS infections
There were 174 cases of proven CNS infections in infants < 90 days old, of which 111 (64%) were viral in origin. One case was excluded due to coinfection with group B streptococcus and EV. EV was the most common identi ed viral pathogen (N = 103; 93%) followed by HSV (N = 7; 6%) and human parechovirus (HPeV) (N = 1; 1%). The HSV cases included 3 with HSV1 (1 with isolated CNS disease, and 2 with disseminated disease) and 4 with HSV2 (1 with isolated CNS disease, and 3 with disseminated disease).

Demographics
The median birth weight was 3345 g (range 1690-4900 g) and median gestational age was 37 weeks (range 29-40 weeks). Infants presented at a median age of 23 (range 3-84) days, with 5 cases occurring during the birth hospitalization (all were EV infection on day 3 to day 21 of life in infants born at 31 to 35 weeks GA). HSV cases presented earlier than non-HSV cases (median 14 days versus 25 days of life; p = 0.02) ( Table 1). Although the vast majority of EV cases occurred during the second half of the year, cases occurred year-round. pneumonitis (N = 1), transaminitis and vesicles (N = 1) and transaminitis, pneumonitis and coagulopathy (N = 2). Coagulopathy was complicated by spontaneous intracranial haemorrhages (intraventricular and parenchymal) in one of these two infants. Extra-CNS manifestations in EV cases included rash (N = 2), pneumonia (N = 2), shock with coagulopathy (N = 2), myocarditis (N = 2) and transaminitis (N = 1). The median age of onset of the 7 cases with organ involvement (omitting the 2 with skin involvement) was 9 days (range 5-73 days). The single HPeV case was not documented to have seizures or extra-CNS disease.

Microbiology
All cases were diagnosed using PCR analysis of CSF. HSV PCR testing was also positive on skin lesions in two infants and from the conjunctiva of one (in the absence of ophthalmological abnormalities). EV and HPeV typing was not available. Suspected urinary tract coinfections occurred in 4 infants with EV infection (Table 1). Systemic candidiasis complicated the course of one infant with HSV encephalitis with liver failure, coagulopathy and intraventricular haemorrhages requiring external ventricular drain placement. Candida albicans was isolated from blood and CSF. Newborn screen, immunoglobulin assay and ow cytometry failed to identify an underlying immunode ciency in this fatal case.

Encephalitis
Thirteen infants (HSV = 6; EV = 7) ful lled criteria for encephalitis. The EV cases presented at a median of 10 days of age (range 5 to 84 days). One case of disseminated HSV2 infection did not meet our de nition of encephalitis as the infant did not have documented seizures and only had a normal head ultrasound documented, but did not have MRI or CT imaging performed. Infants with encephalitis were younger (p = 0.012), more likely to require ICU admission (p < 0.001), more likely to have disseminated disease (p = 0.007) and were more likely to die or have developmental delay (8 (62%) vs 4 (4%); P < 0.001 than those without encephalitis.

Antiviral Treatment and Prophylaxis
All HSV cases received acyclovir treatment for a median of 21 days (range 21-51 days). One infant received acyclovir until demise on day 42 of acyclovir therapy and had persistent PCR positivity in CSF. Pre-mortem CSF resistance testing returned following demise indicating that the HSV was resistant to acyclovir. Another infant did not have documented CSF clearance until 51 days of therapy. For the other 5 cases, repeat testing done between 19 and 22 days of treatment con rmed successful clearance of HSV from CSF. Three (50%) surviving infants were documented to have been discharged on oral acyclovir as prophylaxis for minimum 6 months.

Outcome
There were 2 deaths (2%), one from disseminated EV (a 6-day old infant with myocarditis who required extracorporeal membrane oxygenation) and one from HSV2 (the infant with persistent HSV detection in CSF until death at day 48 of life). Autopsies were not performed on either infant. Virologically-proven recurrence of HSV1 encephalitis presenting as infantile spasms occurred in 1 (33%) of the 3 infants who received oral acyclovir until 6 months of life; this occurred 2 weeks after oral acyclovir was discontinued.

Factors associated with unfavorable outcome
In the univariate analysis, the factors associated with unfavorable outcome included younger age (p = 0.003), seizures (p < 0.001), ICU admission (p < 0.001), a low CSF glucose (p = 0.03), encephalitis (p < 0.001), HSV etiology (p = 0.002), abnormal imaging (p = 0.017), and extra-CNS disease (p = 0.005) ( Table 3). In addition to these six factors, CSF protein > 1 g/L (p = 0.02) and ≥ 1 CSF parameter more in keeping with bacterial meningitis (cell count > 1000 × 10 6 /L, Glucose < 2.0 mmol/L and CSF Protein > 1.0 g/L) were associated with unfavorable outcome in infants with EV infections (p = 0.048) ( Table 3 footnote; Table 4). Sample size limited multivariate analysis.    6 These changing trends may re ect the UK adoption of molecular diagnostic screening for viral meningitis resulting in increased detection over conventional viral culture methods which were not consistently applied in earlier years. 7 Further, molecular testing has facilitated the detection of viruses like HPeV3 that will be missed by viral isolation techniques. 7 Consistent with prior literature, in the current study HSV was much more likely than EV to lead to encephalitis, long-term neurodevelopmental morbidity or death. 3, 8−11 However, among the subgroup of EV cases with encephalitis, we observed a similar rate of adverse outcome to that with HSV encephalitis.
Identifying clinical or laboratory markers that distinguish HSV from non-HSV viral infections is vital to ensure that empiric acyclovir is started at presentation in all HSV cases. 4 13 The importance of performing CSF analysis towards the end of treatment course is exempli ed by the two cases with persistent detection of HSV in CSF, which led to continuing intravenous acyclovir beyond the usual 21-day course.
As noted in our fatal HSV2 case, the possibility of acyclovir resistance should be considered in children with persistent detection of the virus in CSF 14−18 . While rare, the possibility of acyclovir resistance needs to be kept in mind as alternative therapy including foscarnet or vidarabine may be of bene t. 14−16 Limitations of our study include the retrospective design, the lack of EV and HPeV typing and the lack of HPeV testing at most study sites. It is likely that other viral etiologies of meningitis or encephalitis will eventually be identi ed. Methods of molecular testing varied by study center. Emerging molecular diagnostic panels may eventually improve diagnosis of CNS infections (https://www.bio redx.com/products/the-lmarray-panels/ lmarrayme/). Infants with mild viral meningitis are not always recognized to have CNS infection. The total number of infants investigated at the 7 centers to yield the 174 with proven CNS infections is not known. Application of the International Encephalitis Consortium de nition of encephalitis 19 to infants may be inaccurate as it can be di cult to determine if they have altered level of consciousness or focal signs and they are less likely than older children to have fever or CSF pleocytosis. An EEG is not always performed. Therefore, we used a simpli ed de nition for encephalitis which was highly dependent upon the decision to perform and the interpretation of head imaging (which was not always obtained) and recognition of seizures so could have missed or over-diagnosed cases. Infants who had coagulopathy or were too systemically ill to have CSF obtained or who died before they had a diagnosis would have been missed. Molecular testing for HSV (and presumably for other viruses) can be falsely negative early in the course of infection. However, the inclusion of only proven cases was deemed to yield the most accurate data. There was inconsistent access to data on neurodevelopment follow-up and this follow-up was not standardized between centers.
Conclusions Viral CNS infections appear to be more common than bacterial infections in the rst 90 days of life. Age < 21 days and presence of seizures or extra-CNS involvement are clues to HSV infection, even in the absence of CSF pleocytosis. However, not all infants with CNS HSV have seizures. Although most infants with non-HSV viral infections have good outcomes, the subset who have seizures and/or abnormal head imaging may have outcomes similar to those of infants with HSV encephalitis and require neurodevelopmental follow-up. Further studies should address the contribution of HPeV to viral CNS infections and explore predictors of long-term morbidity.

Declarations
Ethics approval and consent to participate: Ethics approval was obtained at each site for conduct of this study with the primary approval coming from the Health Research Ethics Board of the University of Alberta (Study number PRO00055909). Parental consent was waived as it was a retrospective chart review.

Consent for publication: Not applicable
Availability of data and material: All data are stored in REDCap. An anonymized version is available from the corresponding author upon reasonable requests.
Competing interests: Joseph Ting is an Associate Editor for BMC Pediatrics.
Funding: No funding was obtained for this study.
Authors' contributions: JR and MB wrote the rst draft of the protocol, designed the case report form and nalized the manuscript. MB performed the data analysis. DP wrote the rst draft of the manuscript. CR, LO, JB1, JB2, SK, AB, JM, AB, JT and AR provided input into the protocol, case report form or manuscript and organized data collection. All authors approved the nal version.