Dxto de Infecciones en El SNC

D ia g no s ti c Test in g o f
N e u ro l o g i c I n f e c t i o n s
Prashanth S. Ramachandran, MBBS, BMedSci, Michael R. Wilson, MD, MAS*
KEYWORDS
Neuroinfectious diseases Meningoencephalitis Meningitis Encephalitis
Metagenomic next-generation sequencing Neurosyphilis Neurocysticercosis
Neuroborreliosis
KEY POINTS
A thorough clinical evaluation of a patient with meningoencephalitis should guide the
physician toward a thoughtful differential diagnosis that will guide the selection of appro-
priate diagnostic tests to rule in or rule out suspected infections.
Knowledge of the role and accuracy of each of the many diagnostic tests for identifying
neurologic infections is crucial for accurate diagnosis.
Multiplex assays, including unbiased metagenomic next-generation sequencing, promise
to increase diagnostic yield in patients with meningitis and encephalitis.
Neuroinfectious diseases are a major cause of morbidity and mortality worldwide and
have a sizable effect on local health care systems and economies.1,2 A timely diag-
nosis and the institution of appropriate management can drastically improve mortality
and morbidity when the precise organism is known.3 In this review, the authors pro-
vide an overview of the current state of diagnostic techniques for neuroinvasive path-
ogens ranging from culture, polymerase chain reaction (PCR), to serology. The authors
then review new diagnostic modalities, including unbiased metagenomic next-
generation sequencing (mNGS). This overview is not meant to provide an exhaustive
list of all possible diagnostics for all possible neuroinvasive pathogens. Instead, the
authors hope that by reviewing the techniques in detail as they apply to particularly
important and/or common neuroinvasive pathogens, including strengths and common
pitfalls associated with each, the reader will be able to more judiciously select the most
efficient and comprehensive diagnostic approach tailored to their particular patient.
Disclosure Statement: Dr Wilson is supported by the Rachleff Foundation, National Institute for
Neurological Disorders and Stroke grant number K08NS096117, and the UCSF Center for Next-
Gen Precision Diagnostics supported by the Sandler and William K. Bowes, Jr. Foundations.
Dr Ramachandran is supported by the Australian Government Research Training Program
Scholarship.
Department of Neurology, UCSF Weill Institute for Neurosciences, University of California, San
Francisco, 675 Nelson Rising Lane, NS212A, Campus Box 3206, San Francisco, CA 94158, USA
* Corresponding author.
E-mail address: [email protected]
Neurol Clin 36 (2018) 687–703

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688 Ramachandran & Wilson
CULTURE
Cerebrospinal fluid (CSF) culture is the gold standard for central nervous system (CNS)
infections and can provide guidance for antimicrobial therapy. Bacterial cultures are
critical in the management of meningitis with varying sensitivities depending on the
causative organism. These range from 97% for Haemophilus influenzae, 87% for
Streptococcus pneumoniae, and 80% for Neisseria meningitidis.4 The timing of antibi-
otics in relation to the acquisition of CSF is crucial. A positive result decreases from
85% before antibiotics, to 73% when obtained less than 4 hours after therapy, 11%
between 4 to 8 hours and 0% after 8 hours.4,5 Listeria monocytogenes causes both
meningitis and rhombencephalitis with associated abscess formation. Cultures are
hampered by slow growth and are insensitive due to a low CSF bacterial load.6,7 Sen-
sitivities vary between studies and range from 55% to 90% and are as low as 41% in
patients with rhombencephalitis.6,8,9 Blood culture performs marginally better in cases
of rhombencephalitis with rates reaching 61%.9
In general, larger CSF volumes improve the sensitivity of culture. However, even
with large volumes of CSF, visualization of acid fast bacilli (AFB) by microscopy is
only 15% sensitive, and Mycobacterium tuberculosis (TB) can take 2 to 4 weeks to cul-
ture with a sensitivity of only 50% to 60%. Therefore, AFB culture cannot be relied on
for time critical TB meningitis diagnoses.10 In addition, an estimated 480,000 people
developed multidrug-resistant TB in 2015.11 Despite its low yield, TB cultures remain
the gold standard for identifying drug sensitivities.
Viral cultures are performed with cell lines, including rhesus monkey kidney, African
green monkey kidney, A549, and MRC-5. The patient sample is added to the culture
medium, and cytopathic changes are observed in positive cases. These changes can
take up to 30 days to appear depending on the virus.12 Shell vial culturing, antigen detec-
tion, and immunofluorescent antibodies to specific viruses have improved this previ-
ously slow turnaround.13 Enteroviruses are the easiest viruses to culture with 75%
sensitivity and a 3- to 8-day test turnaround.14 Other viruses fail to display equivalent re-
sults. Herpes simplex virus (HSV) is only cultured from CSF in less than 5% of HSV en-
cephalitis cases.15 Fortunately, with the advent of advanced molecular techniques, the
need for viral cultures as a diagnostic tool for CNS infections has diminished.16
Fungal cultures can be performed on specific fungal mediums. However, the 3 most
frequent neuroinvasive fungi: Cryptococcus spp, Candida spp, and Aspergillus spp,
can be cultured on standard bacterial mediums with variable sensitivity. When more
rare fungi are being considered, as may be the case with chronic meningitis, specific
culture mediums are required.17
SEROLOGY
Syphilis
Syphilis is caused by the bacteria Treponema pallidum and can manifest with a variety
of neurologic syndromes depending on the duration of infection and the host’s im-
mune status.18 The diagnostic gold standard is rabbit infectivity testing, but this is
expensive and limited to research laboratories. Dark field microscopy is operator
dependent, time consuming, and not routinely used in the diagnosis of neurosyphilis.
Treponemal nucleic acid detection by PCR in CSF is insensitive.19 Antibody testing is
therefore the standard tool for diagnosis and is divided into 2 groups, treponemal and
nontreponemal testing. Treponemal tests include fluorescent treponemal antibody ab-
sorption (FTA-ABS), T pallidum particle agglutination (TP-PA), and enzyme immuno-
assay (EIA).20 These tests detect antibodies to specific antigenic components of
the bacterium. The latter two tests are more sensitive and specific than the older
Diagnostic Testing of Neurologic Infections 689
FTA-ABS test. These are opposed to nontreponemal tests, which detect antibodies to
lipoidal material released from damaged host cells and cardiolipin-like material
released by T pallidum.21,22 The 2 most common nontreponemal tests are the Ve-
nereal Disease Research Laboratory (VDRL) slide test and the Rapid Plasma Reagin
card test, in which reactive sera produce flocculation of the antigenic material.21
Treponemal tests remain positive for life after primary infection and are not used as a
marker for treatment response. Nontreponemal tests are quantitative and are used to
assess treatment response with the expectation that they will either revert to being
negative or at least exhibit a 4-fold reduction in titer after successful treatment. How-
ever, the nontreponemal tests can be falsely negative either as a result of waning anti-
body titers in late latent syphilis, or conversely, as a result of very high antibody titers
that interfere with the formation of the antigen-antibody lattice, called the prozone
phenomenon. If the latter circumstance is suspected in a high-risk patient, the treating
physician can ask the laboratory to dilute the biological sample and repeat the test.
Because both treponemal and nontreponemal tests are susceptible to false positives
and negatives, combined testing is recommended for an accurate diagnosis of syph-
ilis (the syphilis testing algorithm for syphilis screening is outside the scope of this
article).23 A definitive diagnosis of neurosyphilis is based on a clinical syndrome sug-
gestive of neurosyphilis, a positive serum TP-PA, and a positive CSF VDRL. However,
CSF VDRL only has a sensitivity of roughly 70% and therefore cannot exclude neuro-
syphilis.24 False positives can occur with traumatic taps resulting in contamination
from peripheral blood. In the absence of a positive CSF VDRL, a probable diagnosis
of neurosyphilis is made with a CSF white blood cell count >5 mm/mL or a protein
greater than 45 mg/dL.25 Treponemal tests are not routinely performed on the CSF
despite some suggestions that the high sensitivity of the test should rule out syphilis.26
Varicella Zoster Virus

Varicella zoster virus (VZV) is a neurotropic virus that can cause a wide range of syn-
dromes ranging from encephalomyelitis, multifocal polyradiculitis, and cranial neuritis
to a vasculopathy affecting both small and large cerebral arteries leading to unifocal
and multifocal strokes. Symptoms can present after a prolonged duration, and a
rash can occur months before presentation and may not be present at all.27 VZV
serum immunoglobulin M (IgM) appears within 2 to 5 days of symptom onset. Levels
begin to decrease by 3.5 weeks and cannot be detected by 1 year. IgG levels
decrease with time but generally remain positive for life. Therefore, a positive serum
IgM is usually indicative of active infection.28 CSF evaluation displays a pleocytosis
in two-thirds of patients, and the diagnosis is made with CSF VZV serology or PCR.
VZV IgG levels have higher sensitivity in comparison to PCR, 93% versus 30%,
respectively.29 As most adults will have positive VZV IgG in serum, it is important to
assess for a low-serum/CSF ratio to confirm intrathecal production.27,29 CSF VZV
IgM is also supportive of a diagnosis, despite its less robust sensitivity compared
with IgG.30,31 VZV PCR may also be dependent on the time of symptom onset and
the time of CSF acquisition with decreasing sensitivity of PCR after 1 week. Time-
dependent sensitivity is an important consideration given the protracted course and
delayed presentation of most cases.27,29
Flaviviruses
Flaviviruses cause mosquito and tick-borne infections that are endemic to certain re-
gions throughout the world. They can cause meningitis, meningoencephalitis, and
anterior horn cell disease. West Nile virus (WNV) is endemic to Africa and Europe
and arrived in North America in 1999. Viremia is detected as early as 1 to 2 days after
the primary mosquito bite and persists for up to 1 week until the development of IgM
neutralizing antibodies. Viremia is generally absent by the time neurologic symptoms
appear in immunocompetent hosts, whereas immunocompromised patients demon-
strate a prolonged viremia with a delay in antibody production.32 The pathophysiology
of WNV mirrors the yield of laboratory diagnostics. CSF PCR may be helpful very early
in the disease but generally has a low sensitivity (57%).33 IgM capture enzyme-linked
immunosorbent assay (ELISA) in either blood or CSF during the acute phase is the
gold standard for the diagnosis of neuroinvasive WNV and is generally always present
by the time neurologic symptoms manifest. The large IgM pentameter does not cross
the blood-brain barrier, and therefore, its presence in CSF is suggestive of intrathecal
production.34 IgM may be falsely negative in the early phase in immunocompromised
patients who have not yet mounted an antibody response, and PCR or repeat CSF IgM
assay 7 to 10 days into the illness may be more appropriate in this setting.35 Despite
the high sensitivity of ELISA, the assay has poor specificity because of cross-reactivity
with other neuroinvasive flaviviruses (eg, Zika virus, yellow fever virus, St. Louis en-
cephalitis virus, and dengue virus). Confirmatory testing can be performed using pla-
que reduction neutralization testing (PRNT) for WNV and other flaviviruses. It is also
notable that WNV IgM titers can remain positive for up to 1 year in serum and 7 months
in CSF.36 Therefore, demonstration of a 4-fold or greater increase in virus-specific
antibody titer or elevated virus-specific IgG antibodies in the acute or convalescent
serum sample confirms acute infection.
In 2016, Zika virus spread rapidly through South America and led to an increased
incidence of microcephaly, Guillain-Barré syndrome, encephalitis, and myelitis.37
Once Zika virus was recognized as the etiologic agent 2 years after it was first intro-
duced to Brazil,38 pathogen-specific reverse transcription-PCR and Zika virus IgM
serology were used on CSF to detect neuroinvasive disease. Zika virus serology suf-
fered from the same drawbacks as most flavivirus serologies with false positives due
to cross-reactivity. However, the Euroimmun anti–Zika fever IgG and IgM ELISA tests
demonstrated high specificity for the Zika virus.39 The Centers for Disease Control and
Prevention no longer recommends the PRNT in regions with high prevalence of mul-
tiple flaviviruses due to its low accuracy in this setting.40 Instead, patients are tested
for dengue virus and Zika virus on CSF to rule out cross-reactivity.41
Lyme Disease
Lyme disease is a tick-borne illness secondary to the Borrelia burgdorferi sensu lato
group and is endemic to North America, Europe, and Asia. Borrelia burgdorferi sensu
stricto is the main species found in North America, whereas 5 known species are
endemic to Europe. Lyme disease can present with a wide range of neurologic syn-
dromes, including polyradiculitis, multiple cranial neuropathies, myelitis, meningitis,
brainstem encephalitis, and optic neuritis.42 Diagnostic difficulties are encountered
due to poorly performing assays, insensitivity of US assays against European Borrelia
species, delayed serologic response early in the disease, and the inability for serology
to delineate past and active infection. Hence, it is important to conduct tests in pa-
tients with an appropriate history and examination for neuroborreliosis, therefore
increasing the pretest probability and yield from laboratory investigations. Direct iden-
tification of the spirochete is difficult with resultant low sensitivities for cultures and
PCR. A diagnosis is achieved through a 2-tier system with EIA followed by Western
blot. EIA is highly sensitive, and if positive or equivocal, the Western blot is per-
formed.43 If the symptoms have been present for less than 1 month, then IgM and
IgG are assayed. Two reactive bands constitute a positive IgM, and 5 or more out
of 10 possible bands are a positive for IgG. If symptoms have occurred for longer
than 1 month, then only IgG is performed, although like WNV, IgM antibodies to B
burgdorferi can persist for months. IgM alone cannot be used to confirm a diagnosis,
and evidence of seroconversion may be required.43,44
Both the EIA and the Western blot have significant flaws and may soon be super-
seded by newer assays. The current EIA was developed from whole cell sonicates
of cultured B burgdorferi with no specific targeted antigen, which leads to a high de-
gree of cross-reactivity. The Western blot has poor sensitivity; there are no bands that
are more specific for the organism, and multiple antibodies with similar weights may
colocate over the same band.45 Both these tests perform very poorly with 50% sensi-
tivity in early presentations, and serology may take up to 3 to 6 weeks to become pos-
itive.46 Newer serologic tests target specific antigenic proteins, such as C6, on the
“variable major protein–like sequence, expressed,” a cell surface lipoprotein.47,48
These new assays have demonstrated excellent sensitivity and specificity and may
soon replace the Western blot in the 2-tier algorithm.45,49 However, they suffer from
similar issues of poor sensitivity in early disease, the inability to differentiate between
active and past infection and false negatives in immunocompromised patients.44,50,51
A diagnosis of neuroborreliosis is made with a suggestive history and examination
consistent with Lyme disease, positive serum serology, CSF pleocytosis, and evi-
dence of intrathecal antibody production. Most patients will display a CSF pleocytosis
and elevated protein, except in cases of polyneuropathies.52 In early neurologic dis-
ease, elevated intrathecal antibody production is evident in only 75% of patients
but increases to nearly 100% within several months. The IgG index is elevated in
100% of all late neuroborreliosis cases.53,54 The index can remain elevated for several
years after treatment and cannot be used as a marker for follow-up nor clinical activ-
ity.54 Measurement of C6 on CSF has had variable results and sensitivities.55 CXCL13
is a B-cell–attracting chemokine that has a high sensitivity even before detectable
intrathecal antibodies with decreased levels after treatment.56,57 False positives
have also been found with CNS lymphoma, TB meningitis, and neurosyphilis.56,58
Neurocysticercosis
Neurocysticercosis (NCC) is caused by infection with Taenia solium, a pork tapeworm.
NCC is endemic to Central America, South America, Sub-Saharan Africa, and Asia.59
Diagnosis is made on clinical, exposure history, and radiological characteristics with
confirmatory laboratory diagnosis. The lentil lectin glycoprotein enzyme-linked immu-
noelectrotransfer blot (EITB) is a Western blot assay that is considered the test of
choice.60 This assay uses 6 glycoprotein antigens on a strip to detect antibodies to T sol-
ium. Appearance of any of the 6 bands is consistent with a systemic infection by the
parasite.61 In patients with 2 or more noncalcified or enhancing lesions on brain imaging,
serum EITB carries a sensitivity of 98% and 100% specificity for NCC.62 However, the
EITB performs poorly on samples from patients with single lesions (28%) and calcified
lesions. This may be due to a lack of an antigenic response from dead calcified lesions
compared with viable cysts. Serum carries a slightly higher sensitivity than CSF.63
Compared with the EITB, serum ELISA has poor sensitivity (89%) and specificity
(93%) due to cross-reactivity with other helminthic infections.64 This is less problem-
atic in CSF due to fewer non-NCC antigenic components, allowing for a decreased
test threshold and increased sensitivity.63 CSF titers may also be higher in patients
with subarachnoid, intraventricular, or malignant disease. ELISA also fares poorly
with single or calcified brain lesions.64–66
The main drawback of serology is false positives in asymptomatic patients from
endemic regions and an inability to differentiate between active and inactive infection.
Some studies suggest that 40% of positive results in endemic regions are due to
transient antibodies that become undetectable within 1 year.67 For this reason,
caution must be used when assessing patients from endemic regions, and weight
should not be solely placed on serologic testing, but rather the entire clinical and
neuroradiological information should be considered.
ANTIGEN TESTING
Antigen testing involves detection of antigenic proteins specific to a microbial source

by immunologic methods, such as latex particle agglutination, coagglutination, and
ELISA.
Neurocysticercosis
Monoclonal antibody-based antigen testing using ELISA is commonly used for NCC.
Antigen levels are higher in patients with viable parasites, extraparenchymal disease,
as well as the quantity and size of lesions. CSF samples have a higher sensitivity than
serum. Sensitivity is again lower with calcified and single lesions.68 Antigen testing is
used to monitor treatment response because NCC antigen titers should normalize in
successfully treated patients.68–70
Fungal Antigen Testing

Antigen testing is a rapid and accurate test for the diagnosis of Cryptococcus neofor-
mans. This testing is done through latex particle agglutination or enzyme immunoassay.
The test targets the cryptococcal polysaccharide capsule glucuronoxylomannan. The
sensitivity of antigen testing is very high with 99% sensitivity and 97% specificity.71
The introduction of the point of care lateral flow assay has allowed rapid and accurate
diagnosis of Cryptococcus in resource limited settings. The lateral flow assay can be
performed on serum, plasma, and CSF. It takes approximately 15 minutes for a result
and has a higher sensitivity than standard latex particle agglutination.72,73 Antigen titers
decrease rapidly in response to treatment but may not normalize, with persisting low
titers despite negative cultures, CSF normalization, and clinical improvement. Antigen
testing should not be used to assess for cure.74
Galactomannan is a cell wall polysaccharide that is released by Aspergillus species
during growth. Galactomannan antigen testing uses antibodies directed against
b(1r5)-linked galactofuranosyl residues found on the side chains of galactomannan.75
Its use for the detection of invasive aspergillosis in immunocompromised patients has
been extensively studied in serum and recently in CSF with a sensitivity of 88% in the
latter. Specificity is 96% due to cross-reactivity with Trichocomaceae family, Fusarium
spp, and Histoplasma capsulatum.76 Serum false positives can occur from antibiotic
therapy (piperacillin-tazobactam), bacterial infections, blood transfusions, and dial-
ysis. Sensitivity of the assay increases in patients with hematologic malignancy and
severe neutropenia in comparison to solid organ transplant patients and those with
mild immunosuppression.75
1,3-beta-D-Glucan (BDG) is the major cell wall component of most fungal species,
and BDG antigen testing is used as a broad test for detection of fungal pathogens.
Cryptococcus spp do not contain high levels of BDG in their cell walls and therefore
are not detected. BDG antigen testing is helpful for detecting invasive aspergillosis
and candidiasis. Most studies were conducted on serum that displayed 60% to
100% sensitivity with a recommended test cutoff of 60 to 80 pg/mL.77 After a recent
outbreak of fungal meningitis secondary to contaminated intrathecal methylpredniso-
lone,78 studies have suggested that CSF BDG at a cutoff of 138 pg/mL has a 100%
sensitivity and 98% specificity for Aspergillus fumigatus, Exserohilum rostratum,
Cladosporium cladosporioides, Epicoccum nigrum, and with decreasing titers sug-

gestive of an effective treatment response.79,80
POLYMERASE CHAIN REACTION

Herpes Simplex Viruses
Over the last 2 decades, the advent of PCR has revolutionized the diagnosis of infec-
tious diseases. Its ability to detect common viral and bacterial pathogens has made it
the gold standard in clinical diagnostics.81–83 DNA is extracted from a biological sam-
ple and heated to separate the nucleic acid. Oligomeric primers for the organism-
specific sequences are added with DNA polymerase, leading to transcription of new
DNA, which is complementary to the target sequence. This process is repeated mul-
tiple times with each new strand undergoing the same process, leading to exponential
amplification and increasing sensitivity. Labeled nucleotides are added during the final
run to confirm the suspected genomic sequence.84 The diagnosis of herpes simplex
encephalitis (HSE) was revolutionized by the development of a CSF PCR assay.81,85
Before this, diagnosing HSE required a brain biopsy because viral culture had only
5% sensitivity. HSV-1, 2 PCR has a sensitivity of 98% and 94% specificity.86 False
negatives may occur within the first 72 hours or after 7 to 10 days of antiviral treatment.
If high clinical suspicion exists for HSE, then repeat lumbar puncture (LP) and PCR are
required despite an early negative CSF HSV-1, 2 PCR.87 PCR is available for
numerous pathogens, including standard bacterial meningitis pathogens, VZV,
enterovirus, human herpesvirus-6, Epstein-Barr virus, cytomegalovirus, JC virus,
and WNV. Each PCR has different test performance characteristics, so both negative
and positive results have to be interpreted in clinical context.15
Mycobacterium tuberculosis
The Xpert MTB/RIF is a rapid PCR used as the standard molecular test for the diag-
nosis of pulmonary TB. Xpert sensitivity for TB meningitis is approximately 50%
depending on CSF volume and processing technique.88 The Xpert MTB/RIF also al-
lows detection of rifampicin resistance, a key drug in TB antimicrobial regimens.
The new Xpert MTB/RIF Ultra is the next generation of the Xpert MTB/RIF and has
recently been adopted by the World Health Organization as the test of choice for
the diagnosis of TB meningitis.89 Preliminary studies found a sensitivity of w95%
for TB meningitis when compared with Xpert MTB/RIF or TB cultures combined. How-
ever, when tested against the current uniform case definition for TB meningitis, Xpert
MTB/RIF Ultra demonstrated a sensitivity of only 70%.90
Multiplex Polymerase Chain Reaction
Multiplex PCR is a technique in which multiple primers are used allowing detection of
several organisms by a single assay. The FilmArray meningitis and encephalitis panel
is a rapid, multiplex PCR panel that tests for 14 common viral, bacterial, and yeast
pathogens. A recent prospective multicenter trial evaluating the FilmArray displayed
a range in sensitivity of 85% to 100% depending on the organism. However, there
was also a high rate of false positives and several false negatives.91 The US Food
and Drug Administration has approved this multiplex panel, and some hospitals are
using it as a stand-alone test. Conventional agent-specific confirmation testing by
PCR may be more appropriate in some circumstances.
Bacterial and Fungal Polymerase Chain Reaction
The 16s recombinant ribosomal RNA (rRNA) gene is a highly conserved genetic region
that is found in all bacteria. The sequence is approximately 1550 base-pairs long and
contains both hypervariable and conserved regions. Universal primers are used to
complement either end of the conserved region. The hypervariable regions contain
specific signature sequences useful for bacterial identification at a species level.92
Fungal pathogens can be identified using a similar process with universal fungal
primers targeting the ITS1 and ITS4 conserved regions on the 18s and 28s rRNA se-
quences, respectively. The amplified sequences include the variable ITS2 region for
species identification.93 The use of 16s rRNA PCR for bacterial meningitis has been
encouraging. In culture-proven cases of meningitis, 16s rRNA PCR demonstrated a
sensitivity of 94%, a specificity of 94% confirming a bacterial cause, and was positive
in 30% of culture-negative cases.94 In cases of suspected CNS infection with a CSF
pleocytosis greater than 500 cells/mL (to increase likelihood of bacterial pathogens),
universal primers to 16s and 18s had a 65% sensitivity compared with 35% by micro-
scopy and culture. The main reason for discordance was pretreatment with antibiotics
before LP leading to diminished culture results.95
BRAIN BIOPSY
Before the advent of advanced molecular and immunologic testing, brain biopsy
was considered the gold standard for diagnosis for certain encephalitides. Howev-
er, the yield from brain biopsies is moderate, and its ability to identify a clear cause
in encephalitis is poor.96 Indeed, a recent study demonstrated that the most com-
mon initial pathologic diagnosis after biopsy was “encephalitis of unclear origin.”
Despite this, diagnostic yield may be increased with re-review by a neuropatholo-
gist, careful clinical evaluation with appropriate follow-up, and more advance mo-
lecular and immunologic testing.97 Neuropathology review adds to the hypotheses
of what type of encephalitis might be present even if a specific cause is not
identified.
HYPOTHESIS-FREE TESTING
The performance characteristics of a pathogen-specific test are irrelevant if that or-

ganism is not on the treating physician’s differential diagnosis, and thus, the test is
not ordered. The candidate-based diagnostic approach relies on the unrealistic
expectation that a clinician will have complete knowledge of all pathogenic, local mi-
croorganisms and their clinical manifestations. Although certain pathogens are native
and endemic to specific regions, a constant flux of novel and mutated microorganisms
commonly occurs.98 This ever changing microbial landscape is becoming increasingly
evident in the era of globalization and climate change. Migration and travel have led to
rapid spread of pathogens into new regions, increasing the potential for epidemics
and pandemics. Zika virus is a recent example of a virus that spread rapidly, with
2 years elapsing before its neurologic manifestations became apparent.38
Over the last decade, the cost of whole genome sequencing has fallen drastically
and can now be achieved for less than $1000 with the data being generated in a
day rather than the 10 years and $3 billion it took to sequence the first draft of the hu-
man genome. Unbiased mNGS provides a hypothesis-free and agnostic approach to
the diagnosis of infectious meningoencephalitis. Total DNA and RNA are extracted
from a patient’s biological sample (ie, CSF and/or brain biopsy material) and both
host and nonhost nucleic acid are amplified and then sequenced in a massively par-
allel manner with NGS technologies. After human and environmental contaminant se-
quences are computationally filtered out, the remaining sequences are rapidly
matched against publicly available databases to identify the infectious cause.99
Instead of multiple, targeted PCR tests being performed, mNGS allows testing of
thousands of pathogens including novel organisms within a short timeframe.100 mNGS

can identify early outbreaks and the arrival of novel organisms to a region before large
epidemiologic studies can detect definitive trends.101,102 The final promise of mNGS is
to identify previously overlooked neurotropic pathogens that cause meningoenceph-
alitis, thereby gradually discovering the organisms responsible for some of the large
percentage of cases that are deemed as unknown origin.
Potential Drawbacks
As all the nucleic acid within a sample is amplified in the mNGS assay, invariably there
will be amplification of host and environmental contaminant sequences. The latter can
originate from the patient’s skin flora, microbial nucleic acid present in the collection
tube, and laboratory reagents. This significant “background noise,” which frequently
includes many bacterial and fungal species that have pathogenic potential, can
make interpretation difficult. Thus, stringent measures should always be taken during
sequencing library preparation to minimize cross-contamination. A mock sequence li-
brary is created from water samples and is used as a control, thereby characterizing
the environmental and background microbiome.103,104
In addition to computational solutions to mitigate difficulties discriminating between
signal and noise, molecular depletion and enrichment techniques have been devel-
oped. Commercially available human ribosomal and mitochondrial RNA depletion
kits are not useful for CSF samples because of the very low RNA yields (typically pico-
gram quantities). Therefore, depletion of abundant sequences by hybridization (DASH)
is a tool now being utilized to remove unwanted sequences. DASH uses CRISPR (clus-
tered regularly interspaced short palindromic repeats)-Cas9 technology to target hu-
man complementary DNA (cDNA) within an already amplified sequencing library to
reduce background noise in a highly specific and programmable manner that is
completely agnostic to the input sample type and quantity.105
Conversely, VirCapSeq-VERT is a method for enriching viral sequences in metage-
nomic sequencing libraries by up to 10,000-fold. Approximately 2 million oligonucleo-
tide probes that are designed to bind to the coding site of all viral taxa known to infect
vertebrae are hybridized to a cDNA library. Streptavidin magnetic beads are added to
the probes and their associated cDNA components. The beads are magnetically
captured, cDNA removed, followed by posthybridization PCR. However, this method
only enriches for known viral pathogens.106,107
The delay in processing massive amounts of data used to be the bottleneck in the
timely delivery of clinically pertinent information. However, with rapid development in
bioinformatics pipelines, the time required to process these data has been reduced
drastically. Several pipelines currently exist, including Sequence-based Ultrarapid
Pathogen Identification (SURPI), which is a cloud-compatible, open-access, compu-
tational pipeline used for pathogen identification from complex mNGS data.108 SURPI
was tailored for clinical use, and its speed is suited for clinical application where re-
sults are required within hours. The algorithm initially matches the sequence library
against viral and bacterial databases and can process 7 to 50 million reads within
10 to 30 minutes. If this is negative, a comprehensive review of all pathogens in Gen-
Bank is performed within in 1 to 5 hours. The simultaneous development of both
mNGS and bioinformatics has allowed exponential progress in the field of infectious
diagnostics with promise for ongoing advancements.
Clinical Application
The use of research-based mNGS in the sphere of meningoencephalitis gained mo-
mentum after several notable cases and case series.35,104,109–113 Until recently,
astrovirus was considered only as a gastrointestinal infection and was not a standard
test in the investigation of meningoencephalitis. Several recent cases have demon-
strated a divergent genotype of astrovirus (HAstV-VA1/HMO-C-UK1) that has highly
neurotropic characteristics in immunocompromised patients.100,114–116 Given this dis-
covery, it is now recommended that astrovirus should be considered during the
workup of patients with meningoencephalitis.100 These cases demonstrate the ability
of mNGS to discover new neuroinvasive organisms that have not been previously
considered pathogenic.
The first evaluation of a clinically validated CSF mNGS assay whose results are
reportable in the patient medical record has now been conducted. The Precision
Diagnosis of Acute Infectious Diseases study enrolled 204 patients with idiopathic
meningitis, encephalitis, or myelitis at 8 hospitals, and the study results are
currently under review. This and other studies promise to guide clinicians and
health policy experts as they seek to understand the proper context in which
mNGS testing is most appropriate.
SUMMARY
Neuroinfectious diseases continue to play a major role in morbidity and mortality

worldwide, with many emerging or reemerging infections resulting in neurologic
sequelae.117,118 There is a growing need for rapid and accurate diagnostics that can
lead to meaningful results and curb the significant burden of these diseases. Careful
clinical evaluation of the patient coupled with the appropriate laboratory investigations
leads to the correct diagnosis and implementation of appropriate management.
mNGS is a promising new tool as its ability to identify multiple pathogens in a single
test leads to an unbiased and agnostic approach in the diagnosis of infectious dis-
eases. Prospective studies are forthcoming and will help to answer urgent questions
about the overall performance characteristics of mNGS relative to conventional diag-
nostic modalities. As with other direct detection assays, it is likely that CSF mNGS will
be relatively insensitive for detecting pathogens that are traditionally diagnosed with
serology (eg, WNV and syphilis), that are anatomically localized (ie, brain abscess),
or that have very low titers in the CSF.
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D ia g no s ti c Test in g o f

Neurol Clin 36 (2018) 687–703

Varicella Zoster Virus

Antigen testing involves detection of antigenic proteins specific to a microbial source

Fungal Antigen Testing

Cladosporium cladosporioides, Epicoccum nigrum, and with decreasing titers sug-

POLYMERASE CHAIN REACTION

The performance characteristics of a pathogen-specific test are irrelevant if that or-

thousands of pathogens including novel organisms within a short timeframe.100 mNGS

Neuroinfectious diseases continue to play a major role in morbidity and mortality

1. George BP, Schneider EB, Venkatesan A. Encephalitis hospitalization rates and

8. Mailles A, Lecuit M, Goulet V, et al, National Study on Listeriosis Encephalitis

64. Odashima N, Takayanagui O, Figueiredo J, et al. Enzyme linked immunosorbent

80. Lyons J, Thakur K, Lee R, et al. Utility of measuring (1,3)-b-d-glucan in cerebro-

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