Sanne Hoeks

Chapter 5 62 METHODS Subjects Between January 2003 and January 2013 patients with clinical signs of encephalitis and a positive viral specific polymerase chain reaction (PCR) in blood and/or cerebral spinal fluid (CSF), admitted to the neonatal intensive care at the University Medical Centre, Utrecht, the Netherlands, were eligible for the study. Encephalitis was defined as sepsis-like disease (e.g. rash, problems in thermoregulation, apnoea and/or feeding) and neurological symptoms (e.g. lethargy, irritability and/or seizures). Bacterial cultures remained negative. Residual samples of plasma and/or CSF of 17 infants were stored at -80°C until analysis. Neurodevelopmental outcome was assessed at 6, 12 and 24 months. Infants were seen in the follow-up clinic by the neonatologist, physiotherapist and psychologist. A full neurological assessment, as well as the Griffiths Mental Development Scale (GMDS) or Bayley Scales of Infant and Toddler Development (BSID, 2nd or 3rd edition) were obtained by a certified pediatric physiotherapist to assess cognitive and motor development. 21, 22 We defined moderate delay as a GMDS score and BSID cognitive or motor scale scores < −1 to –2 SDS, and severe delay as scale scores < −2 SDS. Serial cranial ultrasound (cUS) was performed, using a high-resolution (5-8 MHz) transducer (Toshiba). Magnetic resonance imaging (MRI) was performed using a 3 tesla Philips scanner (Achieva, Philips Medical Systems, Best, The Netherlands). MRI included sagittal, axial and/ or coronal T1- and T2-weighted images and diffusion weighted images (DWI). Slice thickness varied between 4 and 1.2 mm. According to abnormalities on cerebral imaging and neurodevelopmental outcome, infants were categorized in three groups: normal, uncertain or severe (table 1). Infants with normal imaging results and scores on neurodevelopmental tests within the normal rangewere classified as normal. Infants with extensive white matter lesions but a favorable neurodevelopmental outcome were indicated as uncertain affected. Patients were labelled as severe when patients died or white matter injury was extensive and combined with an adverse neurodevelopmental outcome with epilepsy, cognitive- and motor developmental delay and/or hearing problems. Multiplex immunoassay CSF and EDTA were centrifuged and stored at -80°C until analysis. Measurements of 33 proteins related to inflammation were performed using an in-house developed and validated (ISO9001:2015 certified) multiplex immunoassay based on Luminex technology (xMAP, Luminex corporation Austin TX USA) as described previously. 23 Interleukin (IL)-1RA, IL-1α, IL-1β, IL-6, IL-7, IL-8 (CXCL8), IL-12, IL-13, IL-17, IL-18, IL-23, IL-25, IL-27, IL-29, IL-33, Interferon (IFN)α,