Revised: July 27, 2026
Accepted: August 26, 2026
Published online: September 25, 2026
Processing time: 106 Days and 19.1 Hours
Viral encephalitis requires rapid etiological assessment, yet targeted polymerase chain reaction, serology, culture, and multiplex panels leave many cases un
Core Tip: Metagenomic next-generation sequencing broadens the diagnostic search in viral encephalitis by detecting pathogen nucleic acids without a predefined target. Its highest value is in severe, unexplained, atypical, or immunocompromised cases after urgent targeted testing and empirical treatment have started. It performs less reliably when pathogen burden is low, infection is tissue-restricted, or diagnosis depends mainly on serology. Results therefore require contamination controls, clinical correlation, and confirmatory testing. Metagenomic next-generation sequencing complements rather than replaces polymerase chain reaction, serology, and conventional microbiology.
- Citation: Binay UD, Karakeçili F, Barkay O, Sümer B. Metagenomic next-generation sequencing in the diagnosis of viral encephalitis. World J Virol 2026; 15(3): 124097
- URL: https://www.wjgnet.com/2220-3249/full/v15/i3/124097.htm
- DOI: https://dx.doi.org/10.5501/wjv.124097
Encephalitis is a clinically important central nervous system disorder characterized by inflammation of the brain parenchyma and may follow an acute or subacute course with substantial morbidity and mortality. Clinically, it may present with fever, headache, altered consciousness, behavioral changes, seizures, focal neurological deficits, and signs of meningeal irritation[1-3]. Although encephalitis may result from infectious, post-infectious, autoimmune, paraneoplastic, or toxic-metabolic causes, viral agents account for a considerable proportion of infectious encephalitis cases. Viral encephalitis is a neurological emergency requiring rapid diagnosis and early treatment, because delayed diagnosis and antiviral therapy may lead to permanent neurological sequelae or death, particularly in treatable conditions such as herpes simplex virus (HSV) encephalitis[3-6].
The clinical significance of viral encephalitis is not limited to the risk of mortality during the acute phase. Survivors may develop long-term neurological consequences, including epilepsy, cognitive impairment, memory loss, personality changes, motor deficits, speech disorders, and the need for prolonged rehabilitation. Therefore, the primary objective in the evaluation of encephalitis is not only to recognize inflammatory brain disease but also to identify the underlying etiological agent as early as possible[2,4,5]. Establishing the etiology enables targeted antiviral or supportive treatment, reduces unnecessary antibacterial or antiviral use, facilitates the recognition of opportunistic infections in immunocompromised patients, and allows detection of arboviral or zoonotic agents of public health importance[7,8].
The causative agents of viral encephalitis may vary according to age, immune status, geographic region, season, vector exposure, travel history, vaccination status, and underlying diseases (Table 1). HSV type 1 is one of the leading causes of sporadic fatal encephalitis in adults. HSV type 2 is more commonly encountered in neonates, immunocompromised individuals, or cases presenting with meningoencephalitis. Varicella zoster virus (VZV) may be associated with encephalitis, vasculopathy, and meningoencephalitis, particularly in older adults and immunocompromised patients. En
| 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 |
The International Encephalitis Consortium emphasized the need for a standardized clinical approach to the diagnosis of encephalitis and recommended the combined assessment of criteria such as altered mental status, fever, seizures, focal neurological findings, cerebrospinal fluid (CSF) pleocytosis, neuroimaging abnormalities, and electroencephalography (EEG) abnormalities in patients with suspected encephalitis[1]. Although this approach enables a more systematic definition of encephalitis cases, advanced microbiological testing is often required to establish the etiological diagnosis[12,13].
Conventional laboratory methods used in the diagnosis of viral encephalitis include polymerase chain reaction (PCR), viral culture, serological assays, antigen tests, and multiplex PCR panels, which have become increasingly available in recent years. HSV PCR in CSF is considered one of the most important tests for the diagnosis of viral encephalitis and is recommended in patients with suspected encephalitis. However, HSV PCR may be negative during the first days of illness or in the presence of a low viral load; therefore, if clinical suspicion persists, repeat testing after several days is recommended[1,2,4,11]. The Infectious Diseases Society of America encephalitis guideline recommends performing HSV PCR on CSF samples in patients with encephalitis and repeating the test when the result is negative despite a compatible clinical presentation. The same guideline notes that viral culture of CSF generally has limited diagnostic value[2].
The major advantages of PCR-based methods are their rapid turnaround time, high sensitivity, and pathogen-specific performance. However, these methods are inherently target-directed. This is a major limitation in diseases such as viral encephalitis, where the etiologic spectrum is broad[1,5]. Targeted PCR assays may remain negative in encephalitis caused by rare, emerging, unexpected, or geographically unusual pathogens. Although multiplex PCR panels can evaluate multiple pathogens simultaneously, their coverage is limited, and they cannot detect viruses outside the panel, novel variants, or unexpected agents[10,11].
Serological tests may be particularly important in arboviral encephalitis. Detection of specific immunoglobulin M in CSF or serum contributes to the diagnosis of infections caused by agents such as West Nile virus or Japanese encephalitis virus. However, serological diagnosis also has limitations. The antibody response may not yet have developed during the early phase of disease; immunocompromised patients may mount a weak serological response; cross-reactivity may occur among flaviviruses; and previous infection or vaccination may complicate interpretation. Therefore, serological results should be evaluated together with the clinical presentation, epidemiological exposure, and, when necessary, confirmatory tests[10,11,14].
Although viral culture has historically been an important method in virological diagnosis, its current role in the diagnosis of encephalitis is limited. Some viruses are difficult to grow in culture, the turnaround time is long, and the viral load in CSF may often be insufficient for successful isolation. Consequently, in acute neurological infections, culture has largely fallen behind PCR and other molecular methods in terms of supporting rapid clinical decision-making[1-3].
Another source of diagnostic difficulty relates to the biological characteristics of CSF specimens. In central nervous system infections, the pathogen burden may be low, the infection may remain restricted to the brain parenchyma, or viral nucleic acid may be present in CSF only transiently[1-3]. The timing of sampling, sample volume, efficiency of nucleic acid extraction, and prior administration of antiviral therapy may all influence test sensitivity. In addition, in some cases, the causative agent may be more readily detected in brain tissue, respiratory samples, serum, or stool rather than in CSF. Therefore, a single negative CSF test is not always sufficient to exclude an etiological cause of encephalitis[4,5].
A major problem in clinical practice is that the etiological diagnosis remains undetermined in a substantial proportion of encephalitis cases despite extensive investigations. Cases of encephalitis with unidentified etiology complicate both patient management and epidemiological surveillance. Missing treatable infections, prolonging unnecessary empirical therapy, delayed consideration of noninfectious causes such as autoimmune encephalitis, and failure to recognize novel pathogens with outbreak potential are among the main consequences of this diagnostic uncertainty[3-5,10,11].
In this context, an ideal diagnostic method for viral encephalitis would be expected to cover a broad pathogen spectrum, reduce the need for predefined diagnostic targets, detect low quantities of viral nucleic acid, identify unexpected or novel pathogens, and provide clinically meaningful results within a reasonable timeframe. Metagenomic next-generation sequencing (mNGS) has attracted increasing attention in recent years as an innovative approach that may address these diagnostic needs[15].
mNGS is an advanced molecular diagnostic approach based on the unbiased sequencing of nucleic acids present in a clinical specimen, followed by bioinformatic analysis of the resulting sequence data to identify potential microbial agents within the sample. Whereas most conventional molecular tests include primers, probes, or panels designed for a specific pathogen or pathogen group, mNGS is not restricted to a predefined target. Therefore, mNGS enables the evaluation of genetic material from viral, bacterial, fungal, parasitic, and even rare or unexpected pathogens within the same analytical process[15-17].
mNGS can generally be applied through two main approaches: DNA metagenomic sequencing and RNA metagenomic sequencing. The DNA-based approach is used to detect DNA-containing organisms, including DNA viruses, bacteria, fungi, and parasites. The RNA-based approach is particularly important for the identification of RNA viruses. Because RNA viruses play an important role in viral encephalitis, an approach based solely on DNA sequencing may miss certain viral agents. Therefore, in central nervous system infections, particularly when the causative agent is unknown, combined evaluation of both DNA- and RNA-based metagenomic analyses may increase the diagnostic yield[17-19].
The fundamental principle of mNGS is the sequencing of both host-derived and microbial nucleic acids present in a specimen without prior discrimination. In samples that are normally considered sterile, such as CSF, this approach is theoretically advantageous because pathogen-derived sequences may be easier to detect in the presence of a low microbial background. However, even in CSF samples, sequences derived from human cells and cell-free nucleic acids may constitute the majority of total reads. This may cause the small number of pathogen-derived sequences to be lost within background noise, reduce sensitivity, or necessitate deeper sequencing[18,19].
One of the most important advantages of metagenomic sequencing in the context of viral encephalitis is its ability to evaluate a broad viral spectrum simultaneously, rather than targeting a single virus. Sequences belonging to diverse viral families, including herpesviruses, enteroviruses, flaviviruses, togaviruses, bunyaviruses, arenaviruses, rhabdoviruses, astroviruses, and bornaviruses, can be investigated in the same analysis[20]. This feature is particularly important when clinical findings are nonspecific, neuroimaging is not diagnostic, or the patient’s epidemiological history is unclear[21,22].
mNGS can also be used not only for pathogen detection but also for genomic characterization of the causative agent. When sufficient viral load and sequencing depth are available, identification at the species or genotype level, phylogenetic analysis, investigation of outbreak relationships, and, in some cases, assessment of mutations associated with antiviral resistance may be possible[19,22]. Therefore, mNGS may also be regarded as a technology that bridges clinical diagnosis and public health surveillance[22].
However, for an mNGS result to be clinically meaningful, technical identification of the detected sequences alone is not sufficient. The microbial sequences obtained must be interpreted together with the clinical presentation, specimen type, immune status of the patient, timing of sampling, accompanying laboratory findings, radiological findings, and possible sources of contamination[23]. For example, an environmental viral contaminant detected with a low number of reads may not represent the clinical causative agent. Conversely, a neurotropic virus detected at a low viral load in an immunocompromised patient may be highly clinically significant. Therefore, mNGS is not merely a laboratory technology but a multidisciplinary diagnostic approach that integrates clinical microbiology, infectious diseases, neurology, and bioinformatics expertise[24-27].
The reliability of an mNGS result depends not only on the capacity of the sequencing platform but also on sample quality, nucleic acid extraction, the library preparation method, sequencing depth, the bioinformatic analysis pipeline, and interpretation of the results within the clinical context[15] (Figure 1).
Platform choice affects read length, throughput, run time, error profile, batching, and the bioinformatic workflow. These characteristics can alter analytical sensitivity and turnaround time, but the platform cannot be judged independently of nucleic acid extraction, library preparation, sequencing depth, controls, and reporting thresholds.
Illumina short-read sequencing has the largest clinical evidence base for CSF mNGS and supports high-throughput DNA and RNA workflows with high per-base accuracy[22,28,29]. Its disadvantages include batch-dependent turnaround, short reads, and the need for substantial laboratory and computational infrastructure. Nanopore sequencing generates long reads in real time and can be deployed on portable instruments, creating a plausible route to faster pathogen identification and genome assembly. Its clinical use, however, remains more dependent on local validation, read-level quality control, contamination management, and carefully defined reporting thresholds.
Direct head-to-head evidence is limited. In the systematic review by Qu et al[27], most eligible studies used Illumina instruments, and the number of studies using other platforms was insufficient for a reliable platform subgroup analysis. Current evidence therefore supports choosing a validated end-to-end assay that meets the clinical question and turnaround requirement; it does not establish one platform as universally superior.
mNGS has gained increasing importance in the diagnosis of viral encephalitis, particularly in cases with unexplained etiology, those in which conventional tests fail to establish a diagnosis, or those in which an unexpected pathogen is suspected[24]. The principal role of mNGS in the diagnosis of viral encephalitis is its ability to detect viral nucleic acids present in clinical specimens in an unbiased manner[18,19]. This feature is especially important in patients with negative standard CSF culture, HSV/VZV PCR, enterovirus PCR, arboviral serology, and multiplex PCR panel results. Through a single analysis, mNGS has the potential to evaluate a broad viral spectrum, including herpesviruses, enteroviruses, flaviviruses, togaviruses, bunyaviruses, arenaviruses, rhabdoviruses, astroviruses, and bornaviruses[22,23]. In this respect, mNGS is not merely an “additional test” in the diagnosis of viral encephalitis, but rather a comprehensive molecular approach that can reshape diagnostic thinking.
Nevertheless, mNGS should not be used as a first-line test in every patient. In conditions such as HSV encephalitis, which require rapid treatment and can be reliably investigated using targeted PCR, conventional PCR assays retain their central role. mNGS is best positioned as a complementary, advanced diagnostic tool for selected patients, including those with persistent clinical suspicion despite negative standard testing, immunocompromised hosts, patients with atypical clinical or radiological findings, and cases involving travel, vector exposure, outbreak potential, or suspected rare pathogens[24,25].
CSF is the most commonly used clinical specimen for mNGS in the diagnosis of viral encephalitis[1]. The main reasons for preferring CSF include its relatively easy acquisition by lumbar puncture, its ability to reflect central nervous system pathology, its status as a normally sterile body fluid, and its central role in the routine evaluation of encephalitis. Detection of pathogen-derived nucleic acid in CSF can strongly contribute to the etiological diagnosis when interpreted in the context of an appropriate clinical presentation[2,3].
Several factors determine diagnostic success in CSF mNGS applications. Among these, the timing of sampling is particularly important. In the early phase of viral encephalitis, viral nucleic acid may not yet have reached a detectable level in CSF. In later stages, the development of the immune response, reduction in viral replication, or initiation of antiviral therapy may decrease the viral load. Therefore, the optimal timing of sampling for mNGS varies according to the clinical stage of the disease, the biology of the causative agent, and the patient’s treatment status. In HSV encephalitis, PCR may be negative early in the disease course; similarly, mNGS may also yield a negative result because of a low viral load. Therefore, a negative mNGS result does not definitively exclude viral encephalitis when clinical suspicion remains strong[18,22-25].
The volume of the CSF sample is also important. Because pathogen-derived nucleic acid may be present in very small quantities in low-biomass specimens, obtaining an adequate volume of CSF may improve sensitivity. However, in clinical practice, sample volume may be limited, particularly in pediatric patients, neonates, or patients with suspected increased intracranial pressure[26,27]. This may affect the sensitivity of mNGS. In addition, appropriate storage conditions, rapid processing, and avoidance of repeated freeze-thaw cycles are important for the detection of RNA viruses[27]. One of the major advantages of CSF mNGS is its ability to detect agents that are not included in targeted PCR panels[28]. Standard diagnostic algorithms generally include HSV-1, HSV-2, VZV, enteroviruses, and, in some regions, arboviruses. However, the viral spectrum capable of causing the clinical syndrome is much broader. mNGS may reduce diagnostic delay by revealing unexpected agents. For example, detection of JC virus, human herpesvirus 6 (HHV-6), cytomegalovirus (CMV), or a rare RNA virus in an immunocompromised patient may alter treatment and follow-up strategies. Similarly, identification of a rare arbovirus in a patient with vector exposure is important not only for individual patient management but also from a public health perspective[27,28].
The clinical value of CSF mNGS has increasingly been supported by large prospective studies. Clinical metagenomic studies in patients with meningitis and encephalitis have shown that mNGS can identify causative agents that are not detected by standard tests in selected cases[25]. Although these studies demonstrate that mNGS can provide genuine clinical benefit in the diagnosis of neuroinfections, they also show that sensitivity and specificity may vary depending on specimen type, pathogen burden, laboratory protocol, and bioinformatic analysis pipeline[28,29].
One limitation of mNGS in CSF samples is the abundance of host-derived nucleic acids. In encephalitis, leukocytosis in CSF, cellular destruction, and inflammation may increase the proportion of human DNA/RNA. The predominance of human sequences reduces the relative proportion of pathogen-derived reads among total reads. This may decrease detection sensitivity, particularly in samples with a low viral load. Therefore, some protocols include preprocessing steps aimed at reducing host nucleic acids or enriching viral particles. However, these procedures are not standardized, and if applied inappropriately, they may lead to the loss of certain pathogens.
Contamination must also be considered when interpreting CSF mNGS results. Because mNGS is a highly sensitive method, low-level nucleic acid contamination originating from laboratory reagents, the environment, or sample processing steps may be detected. Therefore, negative controls should be included in every workflow, cross-contamination between samples should be monitored, and the compatibility of the detected agent with the clinical presentation should be assessed. For example, a virus detected with a low number of reads and also present in the negative control is more likely to represent contamination than a true pathogen. Conversely, a large number of specific reads distributed across different regions of the genome provides stronger diagnostic evidence when compatible with the clinical pre
Another important aspect of CSF mNGS is its indirect contribution to the distinction between infectious and noninfectious diagnoses. Clinical syndromes resembling encephalitis may overlap with autoimmune encephalitis, vasculitis, malignancy, toxic-metabolic encephalopathy, or demyelinating diseases. A negative result from a comprehensive infectious workup may support stronger consideration of noninfectious causes in the appropriate clinical context. However, caution is required; a negative mNGS result does not completely exclude infection. Therefore, results should be interpreted together with the patient’s clinical course and other laboratory and radiological findings[25,27,28].
In conclusion, CSF mNGS is a powerful method with the potential to provide broad pathogen screening, detect unexpected agents, and fill the diagnostic gap in cases in which standard tests are negative. However, its diagnostic performance depends on sample quality, timing, viral load, sequencing depth, bioinformatic analysis, and clinical interpretation. Therefore, CSF mNGS should ideally be used not as a replacement for standard tests but as a complementary method that increases diagnostic yield in selected patients[27].
A wide range of viral agents has been identified by mNGS in cases of viral encephalitis (Table 2). These include classical neurotropic viruses, viruses causing opportunistic infections in immunocompromised patients, arboviruses, zoonotic viruses, and viruses that were previously unexpected or rarely reported[28]. This diversity clearly demonstrates the importance of mNGS in the diagnosis of viral encephalitis.
| 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 |
HSV: HSV-1 in particular is one of the leading causes of sporadic fatal viral encephalitis in adults. HSV encephalitis is classically associated with temporal lobe involvement, fever, altered consciousness, seizures, and lymphocytic pleocytosis in CSF. HSV PCR is a rapid and sensitive test that is considered the diagnostic gold standard. Therefore, targeted PCR should be performed as a first-line test when HSV encephalitis is suspected[6-8]. mNGS may contribute to the diagnosis of HSV encephalitis, particularly in atypical cases, when PCR results are negative or equivocal, or when coinfection with another pathogen is possible. However, for treatable agents that require rapid diagnosis, such as HSV, waiting for mNGS results should not delay empirical acyclovir therapy. Therefore, the role of mNGS in HSV infection is primarily complementary and should be considered within a broader etiological evaluation rather than as a primary diagnostic test[29,30].
VZV: VZV may be associated with encephalitis, meningoencephalitis, myelitis, and central nervous system vasculopathy. In VZV infection, the presence of a skin rash may facilitate diagnosis; however, neurological involvement may also occur in the absence of rash, making diagnosis more difficult. Detection of VZV DNA in CSF by PCR contributes to diagnosis, although assessment of anti-VZV antibodies in CSF may be more sensitive in some cases[1,2,4]. mNGS may be helpful in the diagnosis of atypical presentations such as VZV encephalitis or vasculopathy without rash. In addition, in immunocompromised patients, it provides broad pathogen assessment in situations where VZV may coexist with other herpesviruses or opportunistic pathogens[27-30]. However, the clinical significance of an mNGS result for VZV should be evaluated together with the read count, genomic coverage, CSF findings, and the patient’s clinical presentation.
Enteroviruses: Enteroviruses are important causes of viral meningitis and encephalitis, particularly in children and young adults. Although enterovirus infections are usually mild, some strains may cause severe neurological disease, acute flaccid paralysis, brainstem involvement, or systemic infection in neonates. Targeted enterovirus PCR is commonly used in CSF samples[1,2,4]. mNGS may not only detect enteroviruses but, in some cases, also enable typing and genomic characterization. This feature is important for monitoring strains with high neurovirulence, outbreak investigations, and epidemiological surveillance[22-24]. In addition, mNGS may contribute to the diagnosis in cases with negative enterovirus PCR but persistent clinical suspicion of viral neuroinfection, provided that the specimen type and viral load are suitable[27-30].
West Nile virus and other flaviviruses: West Nile virus is a flavivirus transmitted by mosquitoes, particularly during summer and autumn, and may cause a clinical spectrum ranging from asymptomatic infection to severe neuroinvasive disease. Neuroinvasive disease may present as meningitis, encephalitis, or acute flaccid paralysis. Detection of immunoglobulin M in CSF or serum plays an important role in the diagnosis of West Nile virus infection. However, the antibody response may not yet have developed in the early phase, and cross-reactivity among flaviviruses may occur[9-11]. mNGS may provide diagnostic support in West Nile virus and other flavivirus infections, particularly during the early phase or in unexpected geographic regions. However, viremia in flavivirus infections is usually transient and low-level, and viral RNA may not always be detectable in CSF. Therefore, a negative mNGS result does not exclude arboviral encephalitis. Evaluation should be performed together with serology, epidemiological history, and, when necessary, confirmatory neutralization tests. Japanese encephalitis virus, tick-borne encephalitis virus, and other flaviviruses are also important causes of encephalitis in specific geographic regions[22-24]. Travel history, vector exposure, and seasonality are helpful in considering these agents. The advantage of mNGS in this context is its potential to detect viral sequences even when the clinician has not targeted a specific arbovirus in advance[27-30].
Chikungunya virus: Chikungunya virus is generally recognized as an arboviral agent causing fever, arthralgia, and rash. However, neurological complications may occur in neonates, older adults, and patients with comorbidities. Encephalitis, meningoencephalitis, myelitis, and Guillain-Barré syndrome have been reported. Diagnosis of neurological disease associated with chikungunya virus can be made by PCR or serology, although test sensitivity varies according to the stage of infection[10,11]. mNGS may provide diagnostic value in chikungunya-associated encephalitis, particularly in patients returning from outbreak regions, in the presence of atypical neurological findings, or when targeted testing has not been performed. This example illustrates why mNGS is valuable when geographic and epidemiological assumptions are limited[22-24,27-30].
Powassan virus: Powassan virus is a rare tick-borne flavivirus that can cause severe neuroinvasive infection. Clinically, it may present with fever, headache, altered consciousness, seizures, and focal neurological deficits. Mortality and rates of permanent neurological sequelae may be high. Because it is rare and associated with specific geographic regions, Powassan virus is often not included in routine encephalitis panels. mNGS offers an important advantage in detecting rare arboviruses such as Powassan virus. In patients with tick exposure, negative standard tests, and severe encephalitis, mNGS may identify an unexpected etiological agent[31]. This may affect both patient management and regional surveillance activities.
Astrovirus: Astroviruses were long recognized mainly as agents of gastroenteritis. However, in recent years, astroviruses have been shown to be associated with encephalitis and meningoencephalitis, particularly in immunocompromised patients and in some pediatric cases. In conventional diagnostic panels, astroviruses are usually not considered among neurotropic agents. Therefore, astrovirus-associated neuroinfections may easily be missed by standard methods. mNGS has played a notable role in the diagnosis of astrovirus encephalitis. Detection of astrovirus sequences in unexplained encephalitis cases, particularly in immunocompromised patients, demonstrates the capacity of mNGS to reveal novel or unexpected neurotropic pathogens. However, when astrovirus is detected, its clinical significance should be carefully assessed, because distinguishing gastrointestinal infection, viremia, contamination, and true central nervous system invasion requires clinical context[32-34].
Bornavirus: Bornaviruses have been associated with neurological disease in animals and have also emerged as causes of rare but severe encephalitis in humans. Fatal encephalitis cases associated with variegated squirrel bornavirus and Borna disease virus 1 have demonstrated the importance of metagenomic approaches in identifying novel pathogens. Such cases represent unexpected agents that are difficult to detect using conventional diagnostic tests and are often not included in routine clinical suspicion. Bornavirus-associated encephalitis is among the important examples showing that mNGS is valuable not only for diagnosis but also for pathogen discovery[35-38]. These cases indicate that the etiology of encephalitis has not yet been fully elucidated and that unbiased sequencing may reveal previously unrecognized areas of viral neuroinfection.
One of the most remarkable contributions of mNGS in the field of viral encephalitis is its ability to enable the discovery of novel or unexpected pathogens. Because conventional diagnostic methods are designed to detect known pathogens, identification of a new virus or an agent not previously associated with human disease is often not possible. In contrast, because metagenomic sequencing evaluates nucleic acids in a specimen in an unbiased manner, it can reveal viral sequences that show similarity to known references but have not previously been clinically characterized[39-41].
Novel pathogen discovery is particularly important in encephalitis. A substantial proportion of encephalitis cases remain without an etiological diagnosis despite extensive investigations. This undiagnosed group may include rare viruses, geographically unexpected agents, zoonotic spillover events, opportunistic infections in immunocompromised patients, or pathogens that have not yet been identified[42]. mNGS may fill the diagnostic gap in some of these cases and contribute to a better understanding of the true etiological spectrum of the disease.
The advantages provided by mNGS in novel pathogen discovery can be considered under several headings. First, its unbiased approach allows detection of agents that were not clinically suspected in advance. Second, sequencing of viral genomic fragments enables phylogenetic analysis. Thus, the relationship of the detected virus to known viruses, its possible zoonotic origin, and its pattern of spread can be evaluated. Third, in outbreak investigations, links between cases with identical or similar genomic sequences can be demonstrated. Fourth, genomic information is generated for the development of target-specific PCR or serological assays against newly identified pathogens[39-43].
However, claims of novel pathogen discovery require careful validation. Sequence data alone are not sufficient to demonstrate that a novel or unexpected viral sequence detected by mNGS is the true causative agent of disease. The relationship of the detected virus to the clinical specimen, read count, genome coverage, absence from negative controls, confirmation by independent methods, detection in a second specimen when possible, and consistency with disease pathogenesis should be demonstrated. In addition, supporting evidence such as serological response, tissue tropism, immunohistochemical findings, or animal models strengthens causal inference[39-43].
Novel pathogen discovery is also important from a public health perspective. Zoonotic spillover of viruses into humans, changes in vector distribution associated with climate change, increased global travel, and the expanding population of immunocompromised patients are making the etiology of viral encephalitis increasingly complex. Therefore, mNGS may be considered a strategic technology not only for the diagnosis of individual patients but also for epidemiological surveillance, outbreak early-warning systems, and identification of novel neurotropic viruses[44].
Nevertheless, the limitations of mNGS in novel pathogen discovery should not be overlooked. The absence of similar sequences in reference databases may make it difficult to identify highly divergent viruses. The predominance of host nucleic acids may prevent detection of low-abundance viral sequences. Contamination, misclassification, and database errors may lead to misleading results. In addition, detection of a novel agent does not necessarily imply the existence of a treatable target[45]. Despite these limitations, establishing a diagnosis may have important clinical implications, including discontinuation of unnecessary therapies, prognostic assessment, family counseling, and public health notification.
The largest studies do not support a single, context-free sensitivity estimate for CSF mNGS. In the prospective multicenter study by Wilson et al[22], 58 central nervous system infections were identified in 57 of 204 patients. mNGS detected 13 of these infections only by sequencing (22%); clinicians judged 8 of the 13 results likely to have a clinical effect, and 7 helped guide treatment. The same study also showed the boundaries of the assay: 11 infections were diagnosed only by serology, 7 from tissue or another non-CSF specimen, and 8 were missed because pathogen titers in CSF were low.
Xing et al[25] prospectively evaluated 213 patients and reported an mNGS positivity rate of 57% among definite central nervous system infections, but only 42.6% in definite viral encephalitis or meningitis; at a stringent mapped-read threshold, the area under the receiver operating characteristic curve was 0.659. In contrast, the meta-analysis by Qu et al[27] pooled 12 studies and estimated sensitivity at 77% (95% confidence interval: 70%-82%) and specificity at 96% (95% confidence interval: 93%-98%), with a summary area under the curve of 0.91. Viral-infection subgroup estimates were higher (sensitivity 82%, specificity 97%), yet specificity showed substantial heterogeneity (I2 = 72.07%). These pooled values summarize diverse populations, reference standards, assays, and thresholds and should not be treated as the performance of a single standardized test.
Seven-year clinical experience further clarifies the incremental role of the assay. Benoit et al[28] analyzed 4828 CSF samples; 14.4% were positive for at least one organism. In the adjudicated subset, mNGS alone identified 48 of 220 infections (21.8%). Overall sensitivity was 63.1%, specificity 99.6%, and accuracy 92.9%; sensitivity increased to 86% when the comparator was limited to direct detection in CSF. Across these studies, the recurring signal is high specificity and clinically meaningful incremental diagnoses, accompanied by incomplete sensitivity and marked dependence on how the reference diagnosis is defined (Table 3).
| 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 |
Case mix and reference standards are major sources of variation. A referral cohort enriched for diagnostically difficult or immunocompromised patients differs from an unselected encephalitis cohort, and a composite diagnosis that includes serology or brain tissue will classify more CSF mNGS results as false negative than a CSF-only molecular comparator. Selection bias, retrospective adjudication, and incorporation of sequencing findings into the final diagnosis can also inflate or reduce reported performance[22,25,27,28].
Specimen biology is equally important. Viral load in CSF changes with disease stage and treatment, and some neuroinvasive infections are diagnosed more reliably by intrathecal antibody, serum, respiratory material, stool, or brain tissue. Small sample volume, delayed processing, freeze-thaw cycles, inefficient extraction, host-nucleic-acid excess, and a DNA-only workflow can reduce recovery. Conversely, deeper sequencing may improve detection but also raises cost and increases the number of low-level environmental or reagent signals that require interpretation[18,19,22,29].
Analytical thresholds, database composition, background models, and negative-control strategy vary between laboratories. A permissive threshold may increase sensitivity at the expense of false-positive or clinically irrelevant detections, whereas a stringent threshold can miss low-titer infection. Reporting read count without genomic coverage, control comparison, and orthogonal confirmation obscures this trade-off. Standardized pre-analytical procedures, external quality assessment, transparent thresholds, and prospective blinded adjudication are therefore prerequisites for meaningful study comparison[17,19,27,29].
mNGS adds most value when severe or progressive encephalitis remains unexplained after urgent targeted tests, when the host is immunocompromised, when clinical or radiological findings are atypical, or when travel, vector, zoonotic, or outbreak exposure broadens the differential diagnosis. It can also reveal coinfection or an unexpected pathogen that would not have been included in a fixed panel. It should not delay empirical acyclovir or replace rapid HSV PCR, targeted serology for antibody-defined arboviral disease, testing of a more informative non-CSF specimen, or brain biopsy when tissue-restricted disease remains likely. Low-read detections of latent, reactivated, or contaminating viruses may be non-causal, and a negative result cannot rule out infection. The assay is most useful as a carefully timed diagnostic escalation rather than a universal screen[22-25,27,28].
mNGS broadens the diagnostic search beyond the predefined targets of PCR, serology, culture, and multiplex panels. This benefit is most relevant in selected unresolved cases and should be weighed against the assay’s cost, turnaround time, and interpretive burden[27,29].
A single mNGS analysis can survey a broad pathogen spectrum. This is particularly useful when the phenotype is nonspecific, exposure history is incomplete, or immune suppression makes unusual and mixed infections plausible.
Unlike a targeted molecular assay, mNGS can detect an organism that was not considered when testing was ordered[27,29,45]. This capability matters most for rare, emerging, geographically unexpected, or phenotypically atypical infections[40,41]. The unbiased nature of mNGS is also important in the context of outbreaks and novel pathogens. A specific PCR assay may not be available at the beginning of the emergence of a new virus. In such situations, metagenomic sequencing may contribute to the identification of viral sequences in the specimen, the development of new diagnostic assays, and determination of the outbreak agent. Therefore, mNGS is a strategic technology not only for individual patient diagnosis but also for public health and infectious disease surveillance[44,45].
Novel-pathogen discovery is a distinctive but uncommon application of mNGS. A credible discovery requires independent confirmation and may prompt investigation of zoonotic sources, vectors, transmission routes, and related cases; thus, an individual diagnostic sample can also generate public health evidence.
Another important advantage of mNGS is its ability to detect coinfections. Careful interpretation is essential in the detection of coinfections. In particular, some viruses belonging to the herpesvirus family may be detected at low levels as latent infection or reactivation. This does not always indicate active central nervous system infection. Therefore, the advantage of mNGS in detecting coinfections is not limited to the technical demonstration of more than one microorganism; its true value lies in the clinically meaningful interpretation of these findings within the appropriate context[27].
One of the important strengths of mNGS is its ability to generate multidimensional information from a single clinical specimen. Whereas conventional tests usually provide only a “positive” or “negative” result, mNGS can provide information on the species of the causative agent, read count, genomic coverage, possible variant features, and, in some cases, phylogenetic relationships. These data may be valuable for confirming the diagnosis and for clinical interpretation. For example, detection of only a few reads from a virus does not have the same clinical meaning as detection of numerous reads distributed across different regions of the genome. Including these details in the mNGS report facilitates assessment of result reliability. In addition, when sufficient data are available, more detailed analysis of the viral genome can be used for outbreak investigations or monitoring of novel variants[46]. This multidimensional information makes mNGS not only a diagnostic test but also a tool for clinical research and translational medicine. In complex diseases such as viral encephalitis, it is important not only to identify the causative agent by name but also to understand the genomic characteristics of the pathogen and its relationship with disease[27,47].
Broad coverage and target-independent detection do not make mNGS a universal diagnostic test. Its clinical value depends on understanding where the assay loses sensitivity, where it generates background signals, and how those limitations affect the diagnostic pathway. A negative mNGS result does not exclude infection, and a positive result does not by itself establish causation. Interpretation must integrate the clinical syndrome, CSF profile, imaging, immune status, epidemiology, controls, and conventional test results[19,23,27].
One of the most important limitations of mNGS in routine clinical practice is its high cost. Sequencing instruments, library preparation kits, nucleic acid extraction systems, quality control reagents, bioinformatic infrastructure, data storage, trained personnel, and confirmatory tests all increase the overall cost. Particularly in low- and middle-income countries, this cost is one of the main factors limiting the widespread use of mNGS[19,23,27].
One of the most complex aspects of mNGS is bioinformatic data analysis. Sequencing produces millions of short or long reads. These reads must undergo quality control; adapter sequences and low-quality reads must be removed; human genome-derived sequences must be subtracted; microbial sequences must be compared with reference databases; and potential pathogens must be interpreted from a clinical perspective. This process is much more complex than standard PCR testing[19,23].
Differences in bioinformatic analysis pipelines may lead to different results from the same sample. Quality filters, host genome subtraction methods, alignment algorithms, classification thresholds, reference databases, and reporting criteria may vary between laboratories. This creates a standardization problem. A low read count considered positive in one laboratory may be interpreted as contamination or an indeterminate result in another[27].
The quality of reference databases is another important issue. Databases may be incomplete, incorrectly annotated, contaminated with extraneous sequences, or outdated. Because genetic diversity is particularly high among viruses, accurate identification of novel variants or distantly related viruses may be difficult. Some viral sequences may show similarity to other organisms in short and conserved genomic regions, which may result in misclassification[19,23,27].
Another challenge in bioinformatic analysis is the abundance of host-derived sequences. In CSF or brain tissue samples, human DNA/RNA may constitute the majority of total reads. Removal of human sequences is necessary for both patient privacy and microbial analysis. However, this process increases the computational burden and, in some cases, may inadvertently result in the loss of microbial sequences. In addition, processing human genomic data raises ethical and legal responsibilities[19,23,27].
Bioinformatic expertise alone is not sufficient for interpretation of mNGS data. The clinical significance of the detected organism must also be assessed. At this point, collaboration among infectious diseases, neurology, clinical microbiology, and molecular diagnostics teams is essential. Otherwise, results that are technically correct may still be clinically misleading[19,23,27].
The time required for data analysis is also important for clinical use. In patients with severe encephalitis, waiting several days for a diagnosis may reduce clinical benefit. Therefore, routine use of mNGS requires automated, validated, rapid, and user-friendly analysis pipelines. However, increased automation does not eliminate the need for expert interpretation; expert review remains indispensable, particularly in cases with low-level positivity, rare pathogens, or novel agents[47].
The high sensitivity of mNGS also brings the problem of contamination. Because this method can detect very small quantities of nucleic acid sequences, it may identify not only true pathogens but also environmental, laboratory-derived, or reagent-derived contaminants. This is an important issue, particularly in low-biomass specimens such as CSF. When the true microbial burden in a sample is very low, contaminant sequences may become relatively more prominent within the total microbial read pool[47].
Another important factor affecting the sensitivity of mNGS in the diagnosis of viral encephalitis is low viral load and timing of sampling. In central nervous system infections, pathogen burden follows a dynamic course. Even if viral replication has begun in the early phase of disease, the viral load may not yet have reached a detectable level in CSF. In later stages, the immune response may suppress the virus, or antiviral therapy may reduce the amount of viral nucleic acid. Therefore, a specimen obtained at a single time point may not be sufficient to capture the infection[27,47].
mNGS technology is not available in every center. Because it requires sequencing infrastructure, trained personnel, bioinformatic expertise, and quality systems, it is often performed in reference laboratories or academic centers[27,47]. This may create problems related to sample transfer, turnaround time, and accessibility, particularly in encephalitis cases that require urgent diagnosis.
Clinical accessibility is not limited to the availability of instruments. Multidisciplinary teams capable of correctly interpreting mNGS results are required. When infectious diseases specialists, neurologists, clinical microbiologists, molecular biologists, and bioinformaticians do not work together, the true clinical benefit of the method may be reduced. Therefore, wider implementation of mNGS requires not only technical capacity but also human resources and training[19,23,27].
In the future, decreasing costs, development of automated analysis platforms, wider use of portable devices such as nanopore sequencers, and maturation of clinical guidelines may improve accessibility[48]. However, at present, mNGS should still be regarded as an advanced diagnostic method that should be used in selected patients, with an appropriate clinical question, and in experienced centers[45].
These limitations do not diminish the value of mNGS in the diagnosis of viral encephalitis; rather, they emphasize the need for careful and algorithmic use of the method. With appropriate patient selection, correct timing of sampling, standardized laboratory protocols, contamination control, and multidisciplinary interpretation, the diagnostic contribution of mNGS can be substantially increased.
The value of mNGS is determined less by technological breadth alone than by when it is ordered, which specimen is tested, and whether the result can change a clinical decision. In a patient with suspected viral encephalitis, the initial approach should include clinical stabilization, determination of the need for urgent treatment, and rapid collection of essential diagnostic specimens. Encephalitis should be regarded as a neurological emergency. In patients with altered consciousness, seizures, focal neurological findings, fever, behavioral changes, or signs of meningeal irritation, CSF analysis, neuroimaging, EEG, and basic laboratory tests should be planned without delay. Empirical acyclovir therapy should not be delayed until the possibility of HSV encephalitis has been excluded[1-6]. Even if mNGS is considered at this stage, rapid testing for common treatable agents and empirical treatment strategies should not be postponed.
Incorporation of mNGS into the diagnostic algorithm becomes particularly meaningful when first-line tests are negative but clinical suspicion persists. In the standard approach, CSF cell count, protein and glucose levels, Gram staining and culture, HSV-1/2 PCR, VZV PCR, enterovirus PCR, and arboviral serology according to epidemiological context are performed. Depending on the patient’s immune status, age, travel history, season, and clinical findings, additional tests for CMV, Epstein-Barr virus (EBV), HHV-6, JC virus, human immunodeficiency virus, measles, mumps, rabies, or other viral agents may be added[45,47]. Negative results from these tests, persistent diagnostic uncertainty, and a severe or progressive clinical condition constitute strong indications for mNGS (Figure 2).
Early application of the method may shorten the time to diagnosis; however, unnecessary use may increase costs and generate results that are difficult to interpret. Therefore, patient selection for mNGS should be performed carefully[45].
The first important indication is PCR-negative encephalitis. If encephalitis findings persist despite negative tests for key agents such as HSV, VZV, enteroviruses, and regionally common arboviruses, mNGS should be considered. mNGS may provide diagnostic value particularly when CSF pleocytosis, magnetic resonance imaging lesions compatible with encephalitis, or focal/epileptiform abnormalities on EEG are present and the clinical picture supports an infectious process[27,45,47].
The second important indication is immunocompromised patients. The etiological spectrum of encephalitis is broader in patients with solid organ transplantation, hematopoietic stem cell transplantation, hematological malignancy, advanced human immunodeficiency virus infection, primary immunodeficiency, biological agent use, long-term corticosteroid therapy, or chemotherapy. In addition to classical pathogens, CMV, HHV-6, JC virus, EBV, adenovirus, astrovirus, enteroviruses, fungal agents, parasitic infections, and mixed infections may be encountered in these patients. Furthermore, antibody-based tests may be falsely negative because the serological response may be weak. Therefore, mNGS should be considered at an earlier stage in immunocompromised patients[47].
The third indication is neuroinfections of unknown etiology. In patients with severe disease, progressive neurological deterioration, or no identifiable cause despite extensive investigations, mNGS may be valuable for detecting rare or unexpected pathogens. Within this group, priority should be given to patients treated in intensive care units, those with uncontrolled seizures, progressive radiological lesions, or persistent inflammatory markers[19,23,27,45,47].
The fourth indication is epidemiologically unusual situations. In patients with travel history, vector exposure, animal contact, tick or mosquito bites, occupational exposure, lack of vaccination, outbreak suspicion, or return from a specific geographic region, pathogens outside routine panels may be involved. In such situations, mNGS may provide broad diagnostic evaluation when targeted tests cannot be selected or are not available[27,47].
The fifth indication is clinical uncertainty that may alter treatment decisions despite negative tests. For example, a patient may be receiving empirical acyclovir, broad-spectrum antibacterial therapy, and antifungal therapy, while no causative agent has been identified and the clinical course continues to deteriorate. In such a setting, identification of an agent by mNGS may allow narrowing or modification of the treatment regimen or facilitate consideration of nonin
The sixth indication is cases in which brain biopsy is being considered. If CSF tests are negative, focal or progressive lesions are detected on imaging, and differentiation among malignancy, autoimmune disease, and infection cannot be achieved, brain biopsy may be considered. In such cases, performing CSF mNGS before invasive procedures may provide a diagnosis in some patients and reduce the need for biopsy. Conversely, if CSF mNGS is negative but clinical suspicion persists, mNGS of brain tissue or histopathological and microbiological examination may be considered[27,47].
A stepwise diagnostic algorithm can be proposed for the rational use of mNGS in patients with suspected viral encephalitis. This algorithm preserves the importance of rapid conventional tests while positioning mNGS particularly in cases in which diagnostic uncertainty persists.
Clinical assessment and emergency treatment: When encephalitis is suspected, the first step is clinical stabilization and emergency treatment. Airway, breathing, circulation, seizure control, signs of increased intracranial pressure, and the need for intensive care should be evaluated. Empirical acyclovir therapy should be initiated until HSV encephalitis has been excluded. If bacterial meningitis is included in the differential diagnosis, appropriate antibacterial therapy should not be delayed. Age, immune status, travel history, season, tick or mosquito exposure, animal contact, risk of rabies, vaccination status, occupation, sexual history, medication use, immunosuppression, transplantation history, and previous infections should be assessed[1-7]. This information affects both the selection of targeted tests and the interpretation of mNGS results.
Basic diagnostic investigations: If there is no contraindication, a CSF sample should be obtained. CSF cell count, protein, glucose, lactate, Gram staining, culture, and appropriate molecular tests should be performed. For viral encephalitis, HSV-1/2 PCR, VZV PCR, and enterovirus PCR are among the basic tests. According to regional epidemiology, serology and/or PCR for West Nile virus, Japanese encephalitis virus, tick-borne encephalitis virus, or other arboviruses may be planned. Neuroimaging, particularly magnetic resonance imaging, provides important information in the diagnosis of encephalitis. Temporal lobe involvement may suggest HSV; brainstem involvement may be associated with enteroviruses or some arboviruses; and multifocal white matter lesions may raise suspicion for JC virus or other opportunistic infections in immunocompromised patients. EEG is useful for evaluating seizure activity and diffuse or focal encephalopathic abnormalities[1-8].
Evaluation of initial results: If first-line tests establish a diagnosis, treatment should be tailored accordingly. For example, if HSV PCR is positive, acyclovir therapy should be continued; if enterovirus is detected, unnecessary antibacterial therapy may be discontinued; and if arboviral infection is suspected, supportive treatment and public health notification may be considered. If initial tests are negative but clinical suspicion is low, noninfectious causes should be evaluated. Autoimmune encephalitis, acute disseminated encephalomyelitis, vasculitis, malignancy, toxic-metabolic encephalopathy, and epileptic encephalopathy should be considered in the differential diagnosis. However, if the possibility of infectious encephalitis persists, the diagnostic process should be expanded[1-8].
Assessment of the indication for mNGS: CSF mNGS should be strongly considered when one or more of the following conditions are present: (1) Negative HSV/VZV/enterovirus PCR results and negative basic serological tests; (2) Persistent CSF pleocytosis or radiological findings compatible with encephalitis, or the presence of immunosuppression; (3) A severe, progressive clinical course or a condition requiring intensive care; (4) History of travel, vector exposure, animal contact, or zoonotic exposure; (5) Suspicion of an outbreak or an agent of public health importance; (6) Neuroinfection that remains unexplained by conventional tests; and (7) Need for an advanced noninvasive diagnostic method before considering brain biopsy. At this stage, if possible, an adequate volume of CSF stored under appropriate conditions should be used for mNGS. If a previously obtained and frozen CSF sample is available, it may be used for mNGS after assessment of sample quality. Storage conditions are particularly important for the detection of RNA viruses[23,24,26,27,47].
Interpretation of mNGS results: When mNGS yields a positive result, the clinical significance of the detected pathogen must be assessed. The following questions should be considered: (1) Is the detected virus a known neurotropic agent? (2) Are the patient’s clinical findings and imaging results compatible with this agent? (3) Are the read count and genomic coverage sufficient? (4) Was the same agent detected in the negative control? (5) Is the patient immunocompromised, or is latent viral reactivation possible? and (6) Should the result be confirmed by targeted PCR, serology, or a second specimen? If, after this assessment, the mNGS finding is considered highly likely to represent the causative agent, treatment and follow-up should be adjusted accordingly. If the result remains clinically uncertain, confirmatory tests should be performed and alternative diagnoses should be investigated[23,24,26,27,47].
Approach to negative mNGS results: If mNGS is negative, infection should not be considered completely excluded. If clinical suspicion persists, the timing of sampling, viral load, specimen type, RNA stability, and bioinformatic limitations should be taken into account. When necessary, repeat HSV PCR, arboviral serology, additional testing of serum, respiratory, or stool samples, autoimmune encephalitis panels, malignancy screening, or brain biopsy may be planned. A negative mNGS result, particularly when evaluated together with comprehensive conventional testing, may support consideration of noninfectious causes. However, this decision should be made through a multidisciplinary approach[23,24,26,27,47].
The position of mNGS within the diagnostic algorithm may vary according to different clinical scenarios. Therefore, instead of applying a single uniform approach, patient group-specific assessment should be performed.
Immunocompetent adult patient: In an immunocompetent adult patient presenting with acute fever, altered consci
Immunocompromised patient: mNGS should be considered at an earlier stage in immunocompromised patients. This is because the etiological spectrum is broader, clinical findings may be atypical, and serological tests may be insufficient in this population. CMV, HHV-6, JC virus, EBV, adenovirus, enteroviruses, astrovirus, fungal agents, and parasitic pathogens may not always be adequately covered by conventional algorithms. In this group, mNGS provides important advantages for recognizing mixed infections and detecting opportunistic pathogens. Particular caution is required when interpreting positive results, especially in distinguishing latent viral reactivation from clinically significant disease. For example, detection of HHV-6 or EBV alone should not be accepted as the cause of encephalitis; viral load, clinical presentation, immune status, and confirmatory tests should be evaluated together[23,24,26,27,47].
Pediatric patient: The causative agents of viral encephalitis in children may differ from those in adults. Enteroviruses, parechovirus, HSV, VZV, arboviruses, and vaccine-preventable viruses are important in pediatric patients. HSV and enterovirus infections may be severe in neonates. Limited sample volume and nonspecific clinical findings in children may complicate diagnosis. In pediatric encephalitis cases, mNGS may be useful particularly when standard tests are negative, the disease course is severe, or immunodeficiency is suspected. However, interpretation of results in children should take age-specific pathogens, vaccination history, and sample limitations into account[26].
Patient with travel or vector exposure: Travel history, tick or mosquito exposure, residence in or travel to rural areas, animal contact, or return from an outbreak region requires broadening of the diagnostic algorithm. West Nile virus, Japanese encephalitis virus, tick-borne encephalitis virus, Powassan virus, chikungunya virus, dengue virus, rabies virus, and other zoonotic or arboviral agents should be considered in these patients. mNGS is particularly useful in this scenario when targeted testing is limited or when it is uncertain which agent should be selected for testing. However, it should be remembered that viral RNA in CSF or blood may be present only transiently in arboviral infections. Therefore, mNGS should be evaluated together with serological tests[23,24,26,27,47].
Cases with suspected outbreak or public health concern: The occurrence of multiple similar encephalitis cases, seasonal clustering, animal deaths, increased vector activity, or unexpected geographic distribution may raise suspicion of an outbreak. In this situation, mNGS may play an important role in rapid identification and genomic characterization of the causative agent. Detected viral genomes can be used for phylogenetic analysis and may provide early information to public health authorities[49,50]. In such cases, mNGS should be considered not only for individual patient diagnosis but also for surveillance and outbreak control. However, confirmation of results and official notification processes should be conducted in coordination with relevant public health institutions[23,24,26,27,47].
The clinical benefit of mNGS should be measured by its impact on patient management. A positive mNGS result may influence decisions such as initiation of treatment, modification of therapy, discontinuation of unnecessary treatments, implementation of isolation or public health measures, guidance of additional diagnostic testing, or prognostic as
A negative mNGS result may also contribute to clinical decision-making. A comprehensive negative infectious workup may support stronger consideration of autoimmune encephalitis or other inflammatory diseases. However, it should always be remembered that a negative result does not completely exclude infection[15-19].
For mNGS results to be translated into clinical decisions, reports must be understandable. Reports should include the name of the detected pathogen, read count, genomic coverage, comparison with control samples, interpretation of possible contamination, and level of clinical significance. Categories such as “definite pathogen”, “probable pathogen”, and “finding of uncertain clinical significance” may help clinicians in decision-making[15-19].
mNGS is best understood as a high-specificity, broad-range complement to conventional testing, not as a universal replacement. Its most defensible role in viral encephalitis is early diagnostic escalation for severe, progressive, atypical, immunocompromised, or epidemiologically unusual cases that remain unexplained after urgent targeted testing. The evidence consistently shows clinically important incremental diagnoses, but sensitivity remains incomplete and varies with the reference standard, specimen biology, laboratory workflow, and reporting threshold.
For clinical practice, empirical acyclovir and rapid targeted tests should proceed without waiting for mNGS. Adequate CSF should be collected early, and alternative specimens or serology should be used when pathogen biology requires them. Positive results need control comparison, genome-coverage assessment, clinical correlation, and orthogonal confirmation; negative results must not terminate the investigation when suspicion remains high. Earlier use is particularly reasonable in immunocompromised patients, in whom the pathogen spectrum is wider and conventional findings may be atypical.
Future studies should use prospective enrollment, common case definitions, blinded adjudication, standardized pre-analytical procedures, transparent positivity thresholds, and external quality assessment. Head-to-head comparisons of sequencing platforms should measure not only analytical yield but also turnaround time, cost-effectiveness, changes in treatment, avoidance of invasive procedures, and patient outcomes. These steps are necessary to define when mNGS provides enough incremental value to justify routine clinical deployment.
| 1. | Venkatesan A, Tunkel AR, Bloch KC, Lauring AS, Sejvar J, Bitnun A, Stahl JP, Mailles A, Drebot M, Rupprecht CE, Yoder J, Cope JR, Wilson MR, Whitley RJ, Sullivan J, Granerod J, Jones C, Eastwood K, Ward KN, Durrheim DN, Solbrig MV, Guo-Dong L, Glaser CA; International Encephalitis Consortium. Case definitions, diagnostic algorithms, and priorities in encephalitis: consensus statement of the international encephalitis consortium. Clin Infect Dis. 2013;57:1114-1128. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 948] [Cited by in RCA: 855] [Article Influence: 65.8] [Reference Citation Analysis (0)] |
| 2. | Tunkel AR, Glaser CA, Bloch KC, Sejvar JJ, Marra CM, Roos KL, Hartman BJ, Kaplan SL, Scheld WM, Whitley RJ; Infectious Diseases Society of America. The management of encephalitis: clinical practice guidelines by the Infectious Diseases Society of America. Clin Infect Dis. 2008;47:303-327. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 955] [Cited by in RCA: 745] [Article Influence: 41.4] [Reference Citation Analysis (0)] |
| 3. | Granerod J, Ambrose HE, Davies NW, Clewley JP, Walsh AL, Morgan D, Cunningham R, Zuckerman M, Mutton KJ, Solomon T, Ward KN, Lunn MP, Irani SR, Vincent A, Brown DW, Crowcroft NS; UK Health Protection Agency (HPA) Aetiology of Encephalitis Study Group. Causes of encephalitis and differences in their clinical presentations in England: a multicentre, population-based prospective study. Lancet Infect Dis. 2010;10:835-844. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 1127] [Cited by in RCA: 903] [Article Influence: 56.4] [Reference Citation Analysis (0)] |
| 4. | Steiner I, Budka H, Chaudhuri A, Koskiniemi M, Sainio K, Salonen O, Kennedy PG. Viral encephalitis: a review of diagnostic methods and guidelines for management. Eur J Neurol. 2005;12:331-343. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 154] [Cited by in RCA: 128] [Article Influence: 6.1] [Reference Citation Analysis (0)] |
| 5. | Solomon T, Hart IJ, Beeching NJ. Viral encephalitis: a clinician's guide. Pract Neurol. 2007;7:288-305. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 161] [Cited by in RCA: 112] [Article Influence: 5.9] [Reference Citation Analysis (0)] |
| 6. | Bradshaw MJ, Venkatesan A. Herpes Simplex Virus-1 Encephalitis in Adults: Pathophysiology, Diagnosis, and Management. Neurotherapeutics. 2016;13:493-508. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 275] [Cited by in RCA: 333] [Article Influence: 33.3] [Reference Citation Analysis (0)] |
| 7. | Whitley RJ. Herpes simplex encephalitis: adolescents and adults. Antiviral Res. 2006;71:141-148. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 332] [Cited by in RCA: 333] [Article Influence: 16.7] [Reference Citation Analysis (0)] |
| 8. | Gnann JW Jr, Whitley RJ. Herpes Simplex Encephalitis: an Update. Curr Infect Dis Rep. 2017;19:13. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 85] [Cited by in RCA: 129] [Article Influence: 14.3] [Reference Citation Analysis (0)] |
| 9. | Debiasi RL, Tyler KL. West Nile virus meningoencephalitis. Nat Clin Pract Neurol. 2006;2:264-275. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 140] [Cited by in RCA: 128] [Article Influence: 6.4] [Reference Citation Analysis (0)] |
| 10. | Tyler KL. Emerging viral infections of the central nervous system: part 1. Arch Neurol. 2009;66:939-948. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 56] [Cited by in RCA: 62] [Article Influence: 3.6] [Reference Citation Analysis (0)] |
| 11. | Tyler KL. Acute Viral Encephalitis. N Engl J Med. 2018;379:557-566. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 111] [Cited by in RCA: 194] [Article Influence: 24.3] [Reference Citation Analysis (0)] |
| 12. | Mailles A, Stahl JP; Steering Committee and Investigators Group. Infectious encephalitis in france in 2007: a national prospective study. Clin Infect Dis. 2009;49:1838-1847. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 267] [Cited by in RCA: 288] [Article Influence: 18.0] [Reference Citation Analysis (0)] |
| 13. | Glaser CA, Gilliam S, Schnurr D, Forghani B, Honarmand S, Khetsuriani N, Fischer M, Cossen CK, Anderson LJ; California Encephalitis Project, 1998-2000. In search of encephalitis etiologies: diagnostic challenges in the California Encephalitis Project, 1998-2000. Clin Infect Dis. 2003;36:731-742. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 309] [Cited by in RCA: 302] [Article Influence: 13.1] [Reference Citation Analysis (0)] |
| 14. | Vora NM, Holman RC, Mehal JM, Steiner CA, Blanton J, Sejvar J. Burden of encephalitis-associated hospitalizations in the United States, 1998-2010. Neurology. 2014;82:443-451. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 148] [Cited by in RCA: 194] [Article Influence: 16.2] [Reference Citation Analysis (0)] |
| 15. | Chiu CY, Miller SA. Clinical metagenomics. Nat Rev Genet. 2019;20:341-355. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 1324] [Cited by in RCA: 1140] [Article Influence: 162.9] [Reference Citation Analysis (0)] |
| 16. | Gu W, Miller S, Chiu CY. Clinical Metagenomic Next-Generation Sequencing for Pathogen Detection. Annu Rev Pathol. 2019;14:319-338. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 1181] [Cited by in RCA: 1083] [Article Influence: 154.7] [Reference Citation Analysis (1)] |
| 17. | Schlaberg R, Chiu CY, Miller S, Procop GW, Weinstock G; Professional Practice Committee and Committee on Laboratory Practices of the American Society for Microbiology; Microbiology Resource Committee of the College of American Pathologists. Validation of Metagenomic Next-Generation Sequencing Tests for Universal Pathogen Detection. Arch Pathol Lab Med. 2017;141:776-786. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 237] [Cited by in RCA: 421] [Article Influence: 46.8] [Reference Citation Analysis (0)] |
| 18. | Miller S, Chiu C. The Role of Metagenomics and Next-Generation Sequencing in Infectious Disease Diagnosis. Clin Chem. 2021;68:115-124. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 9] [Cited by in RCA: 123] [Article Influence: 24.6] [Reference Citation Analysis (4)] |
| 19. | Simner PJ, Miller S, Carroll KC. Understanding the Promises and Hurdles of Metagenomic Next-Generation Sequencing as a Diagnostic Tool for Infectious Diseases. Clin Infect Dis. 2018;66:778-788. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 673] [Cited by in RCA: 619] [Article Influence: 77.4] [Reference Citation Analysis (1)] |
| 20. | Naccache SN, Federman S, Veeraraghavan N, Zaharia M, Lee D, Samayoa E, Bouquet J, Greninger AL, Luk KC, Enge B, Wadford DA, Messenger SL, Genrich GL, Pellegrino K, Grard G, Leroy E, Schneider BS, Fair JN, Martínez MA, Isa P, Crump JA, DeRisi JL, Sittler T, Hackett J Jr, Miller S, Chiu CY. A cloud-compatible bioinformatics pipeline for ultrarapid pathogen identification from next-generation sequencing of clinical samples. Genome Res. 2014;24:1180-1192. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 361] [Cited by in RCA: 347] [Article Influence: 28.9] [Reference Citation Analysis (0)] |
| 21. | Wilson MR, Naccache SN, Samayoa E, Biagtan M, Bashir H, Yu G, Salamat SM, Somasekar S, Federman S, Miller S, Sokolic R, Garabedian E, Candotti F, Buckley RH, Reed KD, Meyer TL, Seroogy CM, Galloway R, Henderson SL, Gern JE, DeRisi JL, Chiu CY. Actionable diagnosis of neuroleptospirosis by next-generation sequencing. N Engl J Med. 2014;370:2408-2417. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 851] [Cited by in RCA: 709] [Article Influence: 59.1] [Reference Citation Analysis (3)] |
| 22. | Wilson MR, Sample HA, Zorn KC, Arevalo S, Yu G, Neuhaus J, Federman S, Stryke D, Briggs B, Langelier C, Berger A, Douglas V, Josephson SA, Chow FC, Fulton BD, DeRisi JL, Gelfand JM, Naccache SN, Bender J, Dien Bard J, Murkey J, Carlson M, Vespa PM, Vijayan T, Allyn PR, Campeau S, Humphries RM, Klausner JD, Ganzon CD, Memar F, Ocampo NA, Zimmermann LL, Cohen SH, Polage CR, DeBiasi RL, Haller B, Dallas R, Maron G, Hayden R, Messacar K, Dominguez SR, Miller S, Chiu CY. Clinical Metagenomic Sequencing for Diagnosis of Meningitis and Encephalitis. N Engl J Med. 2019;380:2327-2340. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 912] [Cited by in RCA: 808] [Article Influence: 115.4] [Reference Citation Analysis (0)] |
| 23. | Ramachandran PS, Wilson MR. Metagenomics for neurological infections - expanding our imagination. Nat Rev Neurol. 2020;16:547-556. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 157] [Cited by in RCA: 139] [Article Influence: 23.2] [Reference Citation Analysis (0)] |
| 24. | Brown JR, Bharucha T, Breuer J. Encephalitis diagnosis using metagenomics: application of next generation sequencing for undiagnosed cases. J Infect. 2018;76:225-240. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 198] [Cited by in RCA: 185] [Article Influence: 23.1] [Reference Citation Analysis (1)] |
| 25. | Xing XW, Zhang JT, Ma YB, He MW, Yao GE, Wang W, Qi XK, Chen XY, Wu L, Wang XL, Huang YH, Du J, Wang HF, Wang RF, Yang F, Yu SY. Metagenomic Next-Generation Sequencing for Diagnosis of Infectious Encephalitis and Meningitis: A Large, Prospective Case Series of 213 Patients. Front Cell Infect Microbiol. 2020;10:88. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 147] [Cited by in RCA: 136] [Article Influence: 22.7] [Reference Citation Analysis (0)] |
| 26. | Graff K, Dominguez SR, Messacar K. Metagenomic Next-Generation Sequencing for Diagnosis of Pediatric Meningitis and Encephalitis: A Review. J Pediatric Infect Dis Soc. 2021;10:S78-S87. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 10] [Cited by in RCA: 29] [Article Influence: 5.8] [Reference Citation Analysis (0)] |
| 27. | Qu C, Chen Y, Ouyang Y, Huang W, Liu F, Yan L, Lu R, Zeng Y, Liu Z. Metagenomics next-generation sequencing for the diagnosis of central nervous system infection: A systematic review and meta-analysis. Front Neurol. 2022;13:989280. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in RCA: 26] [Reference Citation Analysis (0)] |
| 28. | Benoit P, Brazer N, de Lorenzi-Tognon M, Kelly E, Servellita V, Oseguera M, Nguyen J, Tang J, Omura C, Streithorst J, Hillberg M, Ingebrigtsen D, Zorn K, Wilson MR, Blicharz T, Wong AP, O'Donovan B, Murray B, Miller S, Chiu CY. Seven-year performance of a clinical metagenomic next-generation sequencing test for diagnosis of central nervous system infections. Nat Med. 2024;30:3522-3533. [RCA] [PubMed] [DOI] [Full Text] [Cited by in RCA: 125] [Reference Citation Analysis (0)] |
| 29. | Miller S, Naccache SN, Samayoa E, Messacar K, Arevalo S, Federman S, Stryke D, Pham E, Fung B, Bolosky WJ, Ingebrigtsen D, Lorizio W, Paff SM, Leake JA, Pesano R, DeBiasi R, Dominguez S, Chiu CY. Laboratory validation of a clinical metagenomic sequencing assay for pathogen detection in cerebrospinal fluid. Genome Res. 2019;29:831-842. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 362] [Cited by in RCA: 466] [Article Influence: 66.6] [Reference Citation Analysis (1)] |
| 30. | Guan H, Shen A, Lv X, Yang X, Ren H, Zhao Y, Zhang Y, Gong Y, Ni P, Wu H, Zhu Y, Cui L. Detection of virus in CSF from the cases with meningoencephalitis by next-generation sequencing. J Neurovirol. 2016;22:240-245. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 70] [Cited by in RCA: 98] [Article Influence: 8.9] [Reference Citation Analysis (0)] |
| 31. | Piantadosi A, Kanjilal S, Ganesh V, Khanna A, Hyle EP, Rosand J, Bold T, Metsky HC, Lemieux J, Leone MJ, Freimark L, Matranga CB, Adams G, McGrath G, Zamirpour S, Telford S 3rd, Rosenberg E, Cho T, Frosch MP, Goldberg MB, Mukerji SS, Sabeti PC. Rapid Detection of Powassan Virus in a Patient With Encephalitis by Metagenomic Sequencing. Clin Infect Dis. 2018;66:789-792. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 39] [Cited by in RCA: 41] [Article Influence: 5.1] [Reference Citation Analysis (0)] |
| 32. | Naccache SN, Peggs KS, Mattes FM, Phadke R, Garson JA, Grant P, Samayoa E, Federman S, Miller S, Lunn MP, Gant V, Chiu CY. Diagnosis of neuroinvasive astrovirus infection in an immunocompromised adult with encephalitis by unbiased next-generation sequencing. Clin Infect Dis. 2015;60:919-923. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 213] [Cited by in RCA: 240] [Article Influence: 21.8] [Reference Citation Analysis (0)] |
| 33. | Quan PL, Wagner TA, Briese T, Torgerson TR, Hornig M, Tashmukhamedova A, Firth C, Palacios G, Baisre-De-Leon A, Paddock CD, Hutchison SK, Egholm M, Zaki SR, Goldman JE, Ochs HD, Lipkin WI. Astrovirus encephalitis in boy with X-linked agammaglobulinemia. Emerg Infect Dis. 2010;16:918-925. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 232] [Cited by in RCA: 255] [Article Influence: 15.9] [Reference Citation Analysis (0)] |
| 34. | Brown JR, Morfopoulou S, Hubb J, Emmett WA, Ip W, Shah D, Brooks T, Paine SM, Anderson G, Virasami A, Tong CY, Clark DA, Plagnol V, Jacques TS, Qasim W, Hubank M, Breuer J. Astrovirus VA1/HMO-C: an increasingly recognized neurotropic pathogen in immunocompromised patients. Clin Infect Dis. 2015;60:881-888. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 143] [Cited by in RCA: 172] [Article Influence: 15.6] [Reference Citation Analysis (0)] |
| 35. | Niller HH, Angstwurm K, Rubbenstroth D, Schlottau K, Ebinger A, Giese S, Wunderlich S, Banas B, Forth LF, Hoffmann D, Höper D, Schwemmle M, Tappe D, Schmidt-Chanasit J, Nobach D, Herden C, Brochhausen C, Velez-Char N, Mamilos A, Utpatel K, Evert M, Zoubaa S, Riemenschneider MJ, Ruf V, Herms J, Rieder G, Errath M, Matiasek K, Schlegel J, Liesche-Starnecker F, Neumann B, Fuchs K, Linker RA, Salzberger B, Freilinger T, Gartner L, Wenzel JJ, Reischl U, Jilg W, Gessner A, Jantsch J, Beer M, Schmidt B. Zoonotic spillover infections with Borna disease virus 1 leading to fatal human encephalitis, 1999-2019: an epidemiological investigation. Lancet Infect Dis. 2020;20:467-477. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 49] [Cited by in RCA: 103] [Article Influence: 17.2] [Reference Citation Analysis (0)] |
| 36. | Tappe D, Pörtner K, Frank C, Wilking H, Ebinger A, Herden C, Schulze C, Muntau B, Eggert P, Allartz P, Schuldt G, Schmidt-Chanasit J, Beer M, Rubbenstroth D. Investigation of fatal human Borna disease virus 1 encephalitis outside the previously known area for human cases, Brandenburg, Germany - a case report. BMC Infect Dis. 2021;21:787. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 4] [Cited by in RCA: 29] [Article Influence: 5.8] [Reference Citation Analysis (0)] |
| 37. | Frank C, Wickel J, Brämer D, Matschke J, Ibe R, Gazivoda C, Günther A, Hartmann C, Rehn K, Cadar D, Mayer TE, Pörtner K, Wilking H, Schmidt-Chanasit J, Tappe D. Human Borna disease virus 1 (BoDV-1) encephalitis cases in the north and east of Germany. Emerg Microbes Infect. 2022;11:6-13. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 3] [Cited by in RCA: 27] [Article Influence: 5.4] [Reference Citation Analysis (0)] |
| 38. | Ebinger A, Santos PD, Pfaff F, Dürrwald R, Kolodziejek J, Schlottau K, Ruf V, Liesche-Starnecker F, Ensser A, Korn K, Ulrich R, Fürstenau J, Matiasek K, Hansmann F, Seuberlich T, Nobach D, Müller M, Neubauer-Juric A, Suchowski M, Bauswein M, Niller HH, Schmidt B, Tappe D, Cadar D, Homeier-Bachmann T, Haring VC, Pörtner K, Frank C, Mundhenk L, Hoffmann B, Herms J, Baumgärtner W, Nowotny N, Schlegel J, Ulrich RG, Beer M, Rubbenstroth D. Lethal Borna disease virus 1 infections of humans and animals - in-depth molecular epidemiology and phylogeography. Nat Commun. 2024;15:7908. [RCA] [PubMed] [DOI] [Full Text] [Cited by in RCA: 24] [Reference Citation Analysis (0)] |
| 39. | Morfopoulou S, Mee ET, Connaughton SM, Brown JR, Gilmour K, Chong WK, Duprex WP, Ferguson D, Hubank M, Hutchinson C, Kaliakatsos M, McQuaid S, Paine S, Plagnol V, Ruis C, Virasami A, Zhan H, Jacques TS, Schepelmann S, Qasim W, Breuer J. Deep sequencing reveals persistence of cell-associated mumps vaccine virus in chronic encephalitis. Acta Neuropathol. 2017;133:139-147. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 31] [Cited by in RCA: 40] [Article Influence: 4.4] [Reference Citation Analysis (0)] |
| 40. | Greninger AL, Messacar K, Dunnebacke T, Naccache SN, Federman S, Bouquet J, Mirsky D, Nomura Y, Yagi S, Glaser C, Vollmer M, Press CA, Kleinschmidt-DeMasters BK, Dominguez SR, Chiu CY. Clinical metagenomic identification of Balamuthia mandrillaris encephalitis and assembly of the draft genome: the continuing case for reference genome sequencing. Genome Med. 2015;7:113. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 82] [Cited by in RCA: 95] [Article Influence: 8.6] [Reference Citation Analysis (0)] |
| 41. | Yozwiak NL, Skewes-Cox P, Stenglein MD, Balmaseda A, Harris E, DeRisi JL. Virus identification in unknown tropical febrile illness cases using deep sequencing. PLoS Negl Trop Dis. 2012;6:e1485. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 134] [Cited by in RCA: 120] [Article Influence: 8.6] [Reference Citation Analysis (1)] |
| 42. | Chiu CY. Viral pathogen discovery. Curr Opin Microbiol. 2013;16:468-478. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 151] [Cited by in RCA: 168] [Article Influence: 12.9] [Reference Citation Analysis (0)] |
| 43. | Lipkin WI. The changing face of pathogen discovery and surveillance. Nat Rev Microbiol. 2013;11:133-141. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 119] [Cited by in RCA: 127] [Article Influence: 9.8] [Reference Citation Analysis (0)] |
| 44. | Traversi D, Pulliero A, Izzotti A, Franchitti E, Iacoviello L, Gianfagna F, Gialluisi A, Izzi B, Agodi A, Barchitta M, Calabrò GE, Hoxhaj I, Sassano M, Sbrogiò LG, Del Sole A, Marchiori F, Pitini E, Migliara G, Marzuillo C, De Vito C, Tamburro M, Sammarco ML, Ripabelli G, Villari P, Boccia S. Precision Medicine and Public Health: New Challenges for Effective and Sustainable Health. J Pers Med. 2021;11:135. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 26] [Cited by in RCA: 49] [Article Influence: 9.8] [Reference Citation Analysis (0)] |
| 45. | Greninger AL. The challenge of diagnostic metagenomics. Expert Rev Mol Diagn. 2018;18:605-615. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 85] [Cited by in RCA: 125] [Article Influence: 15.6] [Reference Citation Analysis (0)] |
| 46. | Miller MB, Tang YW. Basic concepts of microarrays and potential applications in clinical microbiology. Clin Microbiol Rev. 2009;22:611-633. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 277] [Cited by in RCA: 207] [Article Influence: 12.9] [Reference Citation Analysis (0)] |
| 47. | Forbes JD, Knox NC, Ronholm J, Pagotto F, Reimer A. Metagenomics: The Next Culture-Independent Game Changer. Front Microbiol. 2017;8:1069. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 221] [Cited by in RCA: 227] [Article Influence: 25.2] [Reference Citation Analysis (0)] |
| 48. | Greninger AL, Naccache SN, Federman S, Yu G, Mbala P, Bres V, Stryke D, Bouquet J, Somasekar S, Linnen JM, Dodd R, Mulembakani P, Schneider BS, Muyembe-Tamfum JJ, Stramer SL, Chiu CY. Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis. Genome Med. 2015;7:99. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 352] [Cited by in RCA: 385] [Article Influence: 35.0] [Reference Citation Analysis (4)] |
| 49. | de Moraes L, Portilho MM, Vrancken B, Van den Broeck F, Santos LA, Cucco M, Tauro LB, Kikuti M, Silva MMO, Campos GS, Reis MG, Barral A, Barral-Netto M, Boaventura VS, Vandamme AM, Theys K, Lemey P, Ribeiro GS, Khouri R. Analyses of Early ZIKV Genomes Are Consistent with Viral Spread from Northeast Brazil to the Americas. Viruses. 2023;15:1236. [RCA] [PubMed] [DOI] [Full Text] [Cited by in RCA: 4] [Reference Citation Analysis (0)] |
| 50. | Quick J, Loman NJ, Duraffour S, Simpson JT, Severi E, Cowley L, Bore JA, Koundouno R, Dudas G, Mikhail A, Ouédraogo N, Afrough B, Bah A, Baum JH, Becker-Ziaja B, Boettcher JP, Cabeza-Cabrerizo M, Camino-Sanchez A, Carter LL, Doerrbecker J, Enkirch T, Dorival IGG, Hetzelt N, Hinzmann J, Holm T, Kafetzopoulou LE, Koropogui M, Kosgey A, Kuisma E, Logue CH, Mazzarelli A, Meisel S, Mertens M, Michel J, Ngabo D, Nitzsche K, Pallash E, Patrono LV, Portmann J, Repits JG, Rickett NY, Sachse A, Singethan K, Vitoriano I, Yemanaberhan RL, Zekeng EG, Trina R, Bello A, Sall AA, Faye O, Faye O, Magassouba N, Williams CV, Amburgey V, Winona L, Davis E, Gerlach J, Washington F, Monteil V, Jourdain M, Bererd M, Camara A, Somlare H, Camara A, Gerard M, Bado G, Baillet B, Delaune D, Nebie KY, Diarra A, Savane Y, Pallawo RB, Gutierrez GJ, Milhano N, Roger I, Williams CJ, Yattara F, Lewandowski K, Taylor J, Rachwal P, Turner D, Pollakis G, Hiscox JA, Matthews DA, O'Shea MK, Johnston AM, Wilson D, Hutley E, Smit E, Di Caro A, Woelfel R, Stoecker K, Fleischmann E, Gabriel M, Weller SA, Koivogui L, Diallo B, Keita S, Rambaut A, Formenty P, Gunther S, Carroll MW. Real-time, portable genome sequencing for Ebola surveillance. Nature. 2016;530:228-232. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 939] [Cited by in RCA: 992] [Article Influence: 99.2] [Reference Citation Analysis (1)] |