Laboratory diagnosis of CNS infections in children due to emerging and re-emerging neurotropic viruses

Abstract

Recent decades have witnessed the emergence and re-emergence of numerous medically important viruses that cause central nervous system (CNS) infections in children, e.g., Zika, West Nile, and enterovirus/parechovirus. Children with immature immune defenses and blood-brain barrier are more vulnerable to viral CNS infections and meningitis than adults. Viral invasion into the CNS causes meningitis, encephalitis, brain imaging abnormalities, and long-term neurodevelopmental sequelae. Rapid and accurate detection of neurotropic viral infections is essential for diagnosing CNS diseases and setting up an appropriate patient management plan. The addition of new molecular assays and next-generation sequencing has broadened diagnostic capabilities for identifying infectious meningitis/encephalitis. However, the expansion of test menu has led to new challenges in selecting appropriate tests and making accurate interpretation of test results. There are unmet gaps in development of rapid, sensitive and specific molecular assays for a growing list of emerging and re-emerging neurotropic viruses. Herein we will discuss the advances and challenges in the laboratory diagnosis of viral CNS infections in children. This review not only sheds light on selection and interpretation of a suitable diagnostic test for emerging/re-emerging neurotropic viruses, but also calls for more research on development and clinical utility study of novel molecular assays. IMPACT: Children with immature immune defenses and blood-brain barrier, especially neonates and infants, are more vulnerable to viral central nervous system infections and meningitis than adults. The addition of new molecular assays and next-generation sequencing has broadened diagnostic capabilities for identifying infectious meningitis and encephalitis. There are unmet gaps in the development of rapid, sensitive and specific molecular assays for a growing list of emerging and re-emerging neurotropic viruses.

Fig. 1

Timeline of emerging and re-emerging viral diseases that have caused central nervous system infections in the past two decades. The year and geographic area of the indicated outbreaks or epidemics of viral diseases are shown ^,,–. EU, European Union; EEA, European Economic Area.

Fig. 2

Children, especially neonates and infants, are more vulnerable to viral CNS infections and meningitis than adults. This vulnerability may be attributed to children’s unique anatomical and physiological features, including, but not limited to, fast cell division, high basal metabolism, high respiratory rates, thin skin, large body surface area, immature blood-brain barrier and immature immune system. Among them, fast cell division, high basal metabolism and respiratory rates as well as thin skin may be responsible for distinct microbiota/virus colonization in non-CNS body sites ^,. Thin skin and large body surface area may enhance the risk of infection. The immature blood-brain barrier and immune system may lead to vulnerability to CNS infections. ATP, adenosine triphosphate.

Fig. 3

Commonly used clinical diagnostic testing methods for emerging and re-emerging viral diseases. (Left panel) To diagnose neurotropic viral infections, viral nucleic acids, antigens, viruses and antibodies (IgM and/or IgG) can be detected as targets in diagnostic microbiology/virology assays. Pros (middle panel) and cons (right panel) of the indicated detection methods are shown. Viral nucleic acids (red panel) can be detected by using nucleic acid amplification tests, Sanger sequencing and metagenomic next-generation sequencing. Viral antigens (cyan panel) can be detected by using immunohistochemistry and antigen enzyme-linked immunosorbent assays (ELISA). Viruses (purple panel) can be detected by viral culture and shell vial culture. Antibodies (IgM and/or IgG; orange panel) can be detected by ELISA and indirect fluorescent antibody assay. Abbreviations: Ab, antibody; Ag, antigen; CSF, cerebrospinal fluid; C_T, cycle threshold; DENV, dengue virus; EV, enterovirus; IFA, indirect fluorescent antibody; MAC, IgM Ab capture; ME, Meningitis/Encephalitis; NAAT, nucleic acid amplification test; NGS, next-generation sequencing; NS1, nonstructural protein 1; PRNT, plaque reduction neutralization tests; RT-PCR, reverse transcription-polymerase chain reaction; TAT, turnaround time; ZIKV, Zika virus; +ive, positive.

Fig. 4

Classification of molecular methods for infectious diseases diagnostics based on number of targets. Nucleic acid amplification tests (NAATs) can be divided into singleplex (one organism target) and multiplex (> 1 organism targets) tests. ^,, (Top panel) Xpert EV Assay (Cepheid) is an example of singleplex NAAT. (Middle panels) Simplexa HSV-1 &2 Direct (DiaSorin Molecular) is an example of duplex (two organism targets) PCR panel. In addition, a molecular panel for the detection of Zika, Chikungunya and dengue virus in CSF was approved by the FDA under emergency use authorization, which is a triplex (three organism targets) PCR assay. BioFire FilmArray Meningitis/Encephalitis Panel detecting 14 CNS pathogens is the only FDA-approved syndromic panel for CNS infection, which uses a closed pouch to perform cell lysis and nested PCR (PCR 1 and 2). (Bottom panel) Metagenomic next-generation sequencing (NGS) assays were developed to aid in pan-pathogen detection from CSF, which consists of total nucleic acid (RNA/DNA) extraction, library prep, NGS and sequence-based identification. Target numbers of metagenomic NGS assay significantly increase and can report > 1000 organism targets in some instances. (Right) With the increase of target numbers from singleplex to multiplex to metagenomic NGS assays, test throughput increases but flexibility decreases. The bigger a panel/assay is, the more organisms they can detect. However, some organisms on the panel but not on the differential are still tested if BioFire FilmArray Meningitis/Encephalitis Panel or a metagenomic NGS assay is performed. Unexpected organisms detected from the BioFire panel or the metagenomic NGS assay may lead to some confusion and challenges in interpretation of test results. Abbreviations: CHIKV, Chikungunya virus; DENV, dengue virus; EV, enteroviruses; HSV, herpes simplex virus; ID, identification; ZIKV, Zika virus.