Metagenomic next-generation sequencing in the diagnosis of viral encephalitis

Table 1 Viral agents associated with encephalitis

Viral agent	Viral family/group	Common clinical context	Main diagnostic methods	Contribution of mNGS
Herpes simplex virus 1	Herpesviridae	Sporadic necrotizing encephalitis in adults, temporal lobe involvement	CSF HSV PCR, MRI, EEG	Complementary evaluation in PCR-negative, equivocal, or atypical cases
Herpes simplex virus 2	Herpesviridae	Neonatal infection, meningoencephalitis, immunosuppression	CSF HSV PCR, serum/CSF tests	Simultaneous detection within broad pathogen screening
Varicella zoster virus	Herpesviridae	Encephalitis, meningoencephalitis, vasculopathy, immunosuppression	CSF VZV PCR, CSF anti-VZV antibodies	Contribution to diagnosis of VZV neuroinfection without rash or with atypical presentation
Enteroviruses	Picornaviridae	Viral meningitis/encephalitis in children, brainstem encephalitis	CSF PCR, respiratory/stool PCR	Typing, genomic characterization, and detection of strains outside standard panels
Parechovirus	Picornaviridae	Sepsis-like illness and encephalitis in neonates and infants	CSF/blood PCR	Diagnostic contribution in cases not covered by conventional panels
West Nile virus	Flaviviridae	Seasonal arboviral encephalitis, neuroinvasive disease in older adults	Serum/CSF IgM, PCR, neutralization tests	Supportive diagnosis in early-stage or geographically unexpected cases
Japanese encephalitis virus	Flaviviridae	Encephalitis in endemic regions, travel-associated infection	CSF/serum IgM, PCR	Detection in travel-associated cases or when targeted testing has not been performed
Tick-borne encephalitis virus	Flaviviridae	Tick exposure, Europe/Asia-associated encephalitis	Serum/CSF IgM, PCR	Contribution in cases with tick exposure and inconclusive PCR/serology
Powassan virus	Flaviviridae	Tick-borne severe encephalitis	Serology, PCR, public health laboratory tests	Detection of rare arboviruses outside routine panels
Chikungunya virus	Togaviridae	Travel-/outbreak-associated fever, arthralgia, rarely encephalitis	PCR, IgM/IgG serology	Diagnostic contribution in atypical cases with neurological complications
Rabies virus	Rhabdoviridae	Fatal encephalitis after animal exposure	Saliva/skin biopsy PCR, serology, antigen tests	May assist in suspected cases not confirmed by conventional tests
JC virus	Polyomaviridae	Progressive multifocal leukoencephalopathy in immunocompromised patients	CSF JC virus PCR, MRI	Detection during broad diagnostic evaluation in immunocompromised patients
Cytomegalovirus	Herpesviridae	Advanced immunosuppression, transplantation, human immunodeficiency virus	CSF/blood CMV PCR	Assessment of opportunistic infection and coinfection
HHV-6	Herpesviridae	Post-transplant encephalitis, limbic encephalitis	CSF HHV-6 PCR, viral load	Requires careful interpretation to distinguish reactivation, chromosomal integration, and true infection
Astrovirus	Astroviridae	Unexplained encephalitis in immunocompromised patients	mNGS, confirmatory PCR	Identification of unexpected neurotropic agents
Bornavirus	Bornaviridae	Rare, severe/fatal encephalitis, zoonotic association	mNGS, PCR, serology, tissue examination	Important contribution to discovery of novel or rare pathogens

mNGS: Metagenomic next-generation sequencing; CSF: Cerebrospinal fluid; HSV: Herpes simplex virus; PCR: Polymerase chain reaction; MRI: Magnetic resonance imaging; EEG: Electroencephalography; VZV: Varicella zoster virus; IgM: Immunoglobulin M; CMV: Cytomegalovirus; HHV-6: Human herpesvirus 6.

Table 2 Representative viral findings detected or investigated by metagenomic next-generation sequencing and their clinical interpretation

Agent identified/investigated by mNGS	Specimen type	Clinical context	Contribution of mNGS	Points to consider in clinical interpretation
HSV-1/HSV-2	CSF	Acute necrotizing encephalitis, temporal lobe involvement	Complementary diagnosis together with targeted PCR or in PCR-negative/equivocal cases	Empirical acyclovir should not be delayed while awaiting mNGS results
VZV	CSF	Encephalitis without rash, vasculopathy, immunosuppression	Detection of atypical VZV neuroinfection	CSF antibody testing may be more sensitive than PCR in some cases
Enterovirus	CSF, stool, respiratory sample	Pediatric encephalitis, brainstem involvement	Detection of the causative agent and genomic typing	Alternative specimens may be useful if CSF viral load is low
West Nile virus	CSF, serum/plasma	Arboviral encephalitis, seasonal neuroinvasive disease	Detection of viral RNA in early infection or unexpected cases	Should be interpreted together with serology
Chikungunya virus	CSF, serum	Travel-/outbreak-associated neurological disease	Diagnosis in atypical cases without targeted testing	Should be supported by clinical and epidemiological history
Powassan virus	CSF, serum	Severe encephalitis after tick exposure	Identification of a rare arbovirus	Confirmation by a public health laboratory may be required
Astrovirus	CSF, brain tissue	Undiagnosed encephalitis in immunocompromised patients	Discovery of a neurotropic agent not included in conventional panels	Contamination, systemic infection, and true CNS invasion should be distinguished
Bornavirus	CSF, brain tissue	Severe/fatal encephalitis, zoonotic exposure	Discovery of a novel/rare pathogen and genomic characterization	Tissue-level confirmation and epidemiological investigation are important
JC virus	CSF	Leukoencephalopathy in immunocompromised patients	Detection of an opportunistic viral agent during broad screening	Should be evaluated together with clinical and MRI findings
HHV-6	CSF	Post-transplant limbic encephalitis	Detection of an opportunistic viral agent or reactivation	Chromosomal integration and latent reactivation should be considered
CMV	CSF, blood	Advanced immunosuppression, transplantation	Assessment of systemic and CNS involvement	Should be interpreted together with blood viral load and clinical presentation
Unknown/novel virus	CSF, brain tissue	Undiagnosed encephalitis, outbreak or zoonotic suspicion	Novel pathogen discovery, phylogenetic analysis	Independent confirmation, negative controls, and epidemiological assessment are required

mNGS: Metagenomic next-generation sequencing; HSV: Herpes simplex virus; CSF: Cerebrospinal fluid; PCR: Polymerase chain reaction; VZV: Varicella zoster virus; MRI: Magnetic resonance imaging; CNS: Central nervous system; HHV-6: Human herpesvirus 6; CMV: Cytomegalovirus.

Table 3 Comparative summary of key clinical evidence on cerebrospinal fluid metagenomic next-generation sequencing for central nervous system infection

Ref.	Population/workflow	Main findings	Key limitations	Clinical interpretation
Wilson et al[22]; prospective, multicenter	204 patients with meningitis/encephalitis; clinical adjudication against conventional testing	58 infections in 57 patients; 13/58 (22%) identified only by mNGS; 8/13 likely clinical effect; 7/13 guided treatment	Missed 11 serology-only, 7 non-CSF/tissue, and 8 low-titer infections	Adds actionable diagnoses but cannot replace serology, tissue testing, or targeted assays
Xing et al[25]; prospective, multicenter	213 patients with suspected infectious encephalitis/meningitis	mNGS positivity 57% in definite CNS infection and 42.6% in definite viral disease; AUC 0.659 at SSRN ≥ 2	Performance depends on threshold, case definition, and pathogen spectrum	Useful in selected unresolved cases; yield is not uniform across disease groups
Qu et al[27]; systematic review and meta-analysis	12 studies; heterogeneous populations, assays, reference standards, and thresholds	Pooled sensitivity 77% (95%CI: 70%-82%); specificity 96% (95%CI: 93%-98%); AUC 0.91; viral subgroup 82%/97%	Specificity heterogeneity I2 = 72.07%; too few non-Illumina studies for platform comparison	Pooled accuracy is promising but should not be applied as a universal assay estimate
Benoit et al[28]; seven-year clinical performance	4828 CSF samples; adjudicated subset included 220 CNS infections	14.4% of samples positive; mNGS alone identified 48/220 infections (21.8%); sensitivity 63.1%, specificity 99.6%, accuracy 92.9%	Sensitivity rose to 86% against CSF direct detection alone, illustrating reference-standard effects	High specificity and meaningful incremental yield, with persistent sensitivity gaps

mNGS: Metagenomic next-generation sequencing; CSF: Cerebrospinal fluid; CNS: Central nervous system; AUC: Area under the receiver operating characteristic curve; SSRN: Strictly mapped read number; CI: Confidence interval.