Neurology: Neuroimmunology & Neuroinflammation ...€¦ · Web view2016/06/13 · In March 2014,...
Transcript of Neurology: Neuroimmunology & Neuroinflammation ...€¦ · Web view2016/06/13 · In March 2014,...
SUPPLEMENTARY INFORMATION (includes supplementary methods, clinical
summaries, and supplementary Figure e-1)
Supplementary Methods
Tissue biopsy processing and sequencing. Tissue for the study was collected under a
research protocol approved by the Johns Hopkins University School of Medicine
Institutional Review Board (IRB NA_00003551). Fresh frozen tissues were obtained from
eight cases and two were from paraffin-processed tissues. Except for the paraffin-
processed tissues, the small amounts of biopsy material were consumed completely by the
library preparation process, leaving no material for additional studies. Fresh frozen tissues
used for RNA isolation were submerged immediately after biopsy in RNALater. DNA or
RNA isolation, library preparation, and sequencing on Illumina MiSeq platform were all
performed in the Johns Hopkins Deep Sequencing and Microarray Core Facility. All samples
were first treated with lyticase to break down fungal cell walls before proceeding to either
DNA or RNA isolation to ensure capture of any potential fungal sequences in the sample.
Biopsy tissue was snap frozen for DNA isolation or preserved in RNALater for RNA
isolation. Both type of tissue were homogenized in 180 ul Y1 (1M sorbitol with 0.1M EDTA
and b-mercaptoethanol) solution and treated with 20ul 1U/ul lyticase at 30°C for 30
minutes with rotation. DNA was then isolated following the QiaAmp DNA mini-protocol,
and RNA was isolated following the Qiagen miRNeasy protocol for total RNA. Library
preparation was performed using either Illumina DNA Nano sample preparation kit or
Illumina TruSeq stranded total RNA kit for DNA samples (depending on the available
quantity of DNA) and Nugen RNAseq library kit for RNA samples.
For each sample, one run (no multiplexing) of an Illumina MiSeq instrument was used for
sequencing, generating up to 30 million reads with read lengths of 150–300 bp
(Supplementary Table 1). The number of reads varied based on the quality of the DNA and
RNA.
Computational analysis. All of the bacterial and viral genomes are complete–the
chromosomes are free of gaps–although the eukaryotes are of necessity draft genomes,
because no eukaryotic pathogen has yet been completely sequenced. The non-human
genomes were masked to remove low-complexity sequences using the NCBI dustmasker
program[11]. In order to identify common vector contaminants, the database also includes
the genome of phiX174, and the UniVec (ftp://ftp.ncbi.nlm.nih.gov/pub/UniVec/UniVec)
and EMVec (ftp://ftp.ebi.edu.au/pub/databases/emvec/emvec.dat) databases of vector
and adapter sequences. (A more sensitive alignment program such as Bowtie2 could have
been used instead of Kraken. Bowtie2 and similar aligners report the best alignment (or the
best k alignments for some small value of k) for each read rather than the species. They are
not able to report the taxonomic assignment of the reads; thus a read matching two distinct
species would yield two matches, and further software would have to be developed to
determine the best taxonomic assignment, which might be at the genus level or above.
Kraken does this automatically. Also, because Kraken does not do a full alignment of each
read, it is significantly faster than Bowtie2. Finally, we note that the number of unclassified
reads in each sample was typically much less than 1% of the total, indicating that Kraken's
sensitivity was always very high.)
To enrich the Kraken report for the presence of infectious agents, several filtering steps
were implemented. First, reads matching known contaminants such as phage phiX174, a
standard spike-in control for Illumina sequencing instruments, were removed. Next, the
post-processing removed reads from the common human commensal bacteria E. coli and P.
acnes and the potential laboratory contaminant S. cerevisiae [12]. After these corrections,
the total percentage of reads from each remaining species was recomputed to produce the
heatmap in Figure 1. Appendix 1 (Table e-1) shows the read length, total read count, and
number of human reads per sample. Tables e-2 and e-3 contains detailed read counts and
percentages for all species for each sample. Appendix 1 include files containing all reads
found in all samples, after removal of human reads and vector contaminants. Appendix 3
contains a file showing the raw Kraken output for patient PT-5.
As complementary approaches, we analyzed the non-human reads (as classified by Kraken)
with MetaPhlAn [13] and DIAMOND[14]. MetaPhlAn uses a marker-gene approach and has
a database with ~17,000 microbial and eukaryotic reference genomes. We employed
version 2.2.0 with the options --bt2_ps sensitive-local --min_alignment_len 100. DIAMOND
is a fast alternative to BLASTX, which we used to search translated protein sequences from
reads that Kraken failed to classify. In no case did these other classifiers produce results
inconsistent with those from Kraken.
CLINICAL SUMMARIES
Cases with a high degree of diagnostic confidence and positive pathogen
identification
Patient PT-8: A patient with osteomyelitis and multiple nodular lung lesions who
presented with multifocal brain and spinal lesions. Pathogen identified:
Mycobacterium tuberculosis.
A 67-year-old woman presented to another hospital with a two month history of subacute
back pain, fevers and exertional dyspnea. She was transferred to our institution for
evaluation of an “epidural abscess.” Diagnostic evaluation revealed numerous intracranial
and spinal cord lesions, multiple lung nodules, lumbar osteomyelitis and epidural abscess
at the site of two previous steroid injections for back pain and bilateral psoas muscle
abscesses. Extensive microbiological studies that included samples from vertebral bone
biopsy, serum, sputum and bronchoalveolar lavage (BAL) fluid and bronchial and lung
biopsies were negative for bacteria, fungi or mycobacteria. Two BAL fluid samples were
also negative for mycobacteria when tested with Xpert MTB/RIF ® assay [1].
QuantiFERON-TB Gold ® interferon-gamma release assays (IGRA) obtained on four
separate occasions during the pre-biopsy clinical course were indeterminate. Two
cerebrospinal fluid samples showed mild pleocytosis (6 leukocytes/mm3 in sample 1 and
15 leukocytes/mm3 in sample 2) and elevated protein (72 mg/dL in sample 1 and 103
mg/dL in sample 2). Most of the CSF microbiological studies which included stains for AFB,
mycobacteria and fungal cultures as well as other assays for fungal or bacterial species
were negative. The patient experienced a rapid worsening of her neurological condition
and became unresponsive, ultimately requiring prolonged intubation. She had been on a
long-standing and broad-spectrum antibiotic treatment but in the context of her
decompensation an anti-tuberculous drug regimen was empirically added. She improved
transiently without clear diagnosis and was reluctant to proceed with any additional
biopsies or tissue. A research assay of CSF using the PLEX-ID platform [2] was positive for
Nocardia spp. Because Nocardia was felt to be a plausible explanation of her multiple brain
and pulmonary nodules, and due to the absence of other potential pathogens in her workup
and her improvement on antibiotics that were active against Nocardia, she was taken off
anti-tuberculous therapy and transitioned to trimethoprim/sulfamethoxazole and
meropenem. Steroid treatment that had been instituted for management of brain and
spinal cord edema was tapered. She was discharged to a rehabilitation facility but later re-
admitted with a worsening neurological condition and encephalopathy. Brain MRI showed
progression of multiple nodular enhancing lesions throughout supratentorial and
infratentorial brain structures compared to prior imaging obtained 4 weeks before (Figure
2). The patient agreed to pursue a brain biopsy for diagnosis. Biopsies from the perilesional
brain tissue (S1) and a nodular lesion (S2) were obtained for conventional pathology,
microbiology, and NGS studies.
Two DNA sequencing runs yielded 15M reads from sample S1 and 14M reads from sample
S2. These runs yielded the fewest microbial reads of any of the patients in our study: 18
and 22 bacterial reads, and only one to six viral and fungal reads, respectively, for samples
S1 and S2. Nonetheless, a clear finding emerged for sample S2: 15 reads from
Mycobacterium tuberculosis. Despite the small absolute number of reads, this species
explained 68% of the bacterial reads detected. We manually confirmed the sequence
assignments using Blast [3] to align them against the NCBI nt database. We then re-aligned
all reads against one specific genome, M. tuberculosis 7199-99 (accession NC_020089.1)
using Bowtie2 with sensitive local alignment settings[4]. This procedure yielded 34 reads
that were randomly distributed along the M. tuberculosis genome. As additional support for
this diagnosis, we note that M. tuberculosis was not observed as a contaminant in any other
sample in this case series, and detection in a brain biopsy due to quiescent infection is
unexpected.
Histopathological studies of the corresponding S2 sample showed necrotizing granulomas
although extensive studies with AFB, GMS and other special stains failed to identify any
microorganism (Figure 2). Because the clinical symptoms of the patient were consistent
with tuberculosis, necrotizing granulomas were present in the biopsy, and M. tuberculosis
was identified by sequencing, treatment for tuberculosis was re-initiated the same day that
sequencing was completed. The patient responded rapidly over the next few days and was
discharged to continue her anti-tuberculous treatment at home. The patient has exhibited
nearly complete cognitive and neurologic recovery, although at 5 months after the
diagnosis was established, continued with residual back pain resulting from her spine
pathology.
Patient PT-5: A patient with a focal lesion in the left hemisphere with inconclusive
CSF findings suspected to be PML. Pathogen identified: JC polyomavirus.
A 52-year-old man was admitted for evaluation of right lower extremity weakness and gait
disturbance which evolved to right hemiparesis. He presented later with a simple partial
motor seizure. The patient had experienced previous episodes of dysarthria. At the time of
his initial symptoms, a brain MRI showed focal atrophy of the left post-central and adjacent
superior frontal gyri and a focal white matter signal abnormality. During the course of
hospitalization his pattern of weakness progressed and he developed focal motor seizures
and complex partial seizures which became resistant to multiple anti-epileptics. A
subsequent brain MRI showed an increase in size of the left frontoparietal-occipital lesion
with mild mass effect (Figure 3A). CSF studies which included PCR for viruses (VZV, HSV,
CMV, EBV, JC) and bacterial cultures were negative. The patient was recommended for a
brain biopsy and transferred to our institution for further evaluation. Prior to biopsy,
repeat CSF PCR studies were again inconclusive or “low DNA levels” for JC polyomavirus.
Interestingly the patient did not have any previously identified immunocompromising
conditions (a common risk factor for JC polyomavirus) and tested negative for HIV and for
autoimmune disorders. The patient underwent a biopsy of the left parietal-occipital lesion
which was processed for NGS as well as neuropathological studies. RNA sequencing
yielded 26.9 million reads, of which 25.9 million were human (Supplementary Table 1).
Analysis found a very strong presence of JC polyomavirus, with 8,944 out of 8,954 reads
from all viruses. Although many bacterial species were detected, JC polyomavirus was the
most abundant species in terms of the number of reads, despite its small genome size. The
whole genome of JC polyomavirus was covered by the reads, at an average depth over 200
(Figure 3B). We therefore concluded that the sequence data showed strong support for
infection with JC polyomavirus, a known cause of PML [5], as suggested by the initial MRI
studies of this patient. Three days after sequencing results were obtained, pathology
results included marked astrogliosis and intra-nuclear inclusions in oligodendrocytes
(Figure 3C) and positive immunostaining for SV40 T antigen (a surrogate for JC
polyomavirus), confirming the diagnosis (Figure 3D). Following the diagnosis of PML and
exclusion of risk factors for immunosuppression such as HIV infection, malignancy and
autoimmune disorders, the only risk factor found was a persistent leukopenia (average
leukocyte count of 2500/mm3). Blood analyses revealed lymphopenia (1200 cells/mm3)
with an absolute number of CD4+ T cells of 352 cells/mm3 and normal percentages of CD8+
and CD3 positive T cells (33.2% and 51.2%, respectively). Interestingly, two post-biopsy
PCR tests in CSF using a well-validated quantitative PCR assay (Ryschkewitsch et. el. J
Clinical Virology 2013), both obtained after worsening of the patient’s clinical status and
brain MRI changes, gave negative results.
Patient PT-10: A patient with multifocal brain lesions and a history of organ
transplants and immunosuppression. Pathogen identified: Epstein-Barr virus.
A 44-year-old woman with previous history of kidney and pancreatic transplant 10 and 8
years previous to the onset of neurological symptoms presented with facial paralysis. Brain
imaging studies showed at least three enhancing lesions in both hemispheres, one of them
had the appearance of a “ring enhancing” lesion which resembled CNS toxoplasmosis
(Figure 4A-C). Assessment of the CSF which included CSF microbiological and PCR analysis
for known opportunistic infections were negative. CSF flow cytometry failed to reveal any
evidence of CNS malignancy such as lymphoma. A brain biopsy from one of the lesions
localized in the left parietal lobe was obtained one month after the onset of symptoms.
Pathology assessment showed granulomatous and lymphohistocytic inflammation with foci
of necrosis (Figure 4D), histopathological features consistent with encephalitic changes
rather than a lymphoproliferative disorder. Flow cytometry studies of the tissue failed to
identify clonal lymphoid populations. Microbiological studies including cultures and special
stains for fungi and bacteria were negative. Paraffin sections were processed for NGS
sequencing, which yielded 21.3 million reads (Table 1), of which 21 million were human
and ~216,000 were vector or synthetic controls. Only 569 reads were bacteria, all
matching known skin bacteria or contaminants. Twenty reads matched viruses, of which 18
(90%) matched Epstein-Barr virus (EBV). As we had not previously observed EBV in any
samples, these reads appeared unlikely to be due to contamination. Based on the NGS
finding, laboratory validation tests using in situ hybridization for EBV-encoded RNA
(EBER) were performed on the brain biopsy. These laboratory tests took approximately ten
days, and the results confirmed EBV infection (Figure 4E). These positive findings were
conveyed to the clinicians and used to adjust immunosuppressive treatment. This case
shows many similarities to a previous report [6] of EBV-induced brain lesions in an
immunosuppressed patient following organ transplantation.
Cases with indeterminate pathogen identification but possible infection
Patient PT-2: A patient with a Tolosa-Hunt-like syndrome with focal
pachymeningitis.
A 69-year-old man was evaluated in the neuroimmunology clinic after referral from his
ophthalmologist and otorhinolaryngologist both of whom had followed him for a history of
left retro-orbital pain and ophthalmoplegia. His symptoms had progressed for two years,
beginning with a sensation of “pressure” behind his eye, left hemicranial headache and
blurred distance vision. He was diagnosed initially with “mild iritis” and given topical
steroid treatment. Cataract surgery was performed in December 2012 (left eye) and
January 2013 (right eye). Both procedures included intraocular lens implantation. Almost
two months after the first procedure, he developed left-sided ptosis and retro-orbital
headache. He was given treatment with Cefdinir as treatment for “sinusitis.” He remained
symptomatic with a variable degree of retro-orbital headache. In March 2014, he
developed left retro-orbital headache, decreased vision, horizontal diplopia,
ophthalmoplegia and facial numbness in the V1 trigeminal nerve distribution. He was
diagnosed with Tolosa-Hunt syndrome and prescribed daily dexamethasone which
resulted in partial improvement of his headache and facial numbness. He was referred to
our institution for a second opinion. A clinical assessment was consistent with left-sided
ptosis and ophthalmoplegia with left IV and VI cranial nerve palsies and residual III nerve
palsy without pupil involvement. A brain MRI demonstrated pachymeningeal and
leptomeningeal enhancement localized in the medial aspect of the left middle cranial fossae
extending to the orbital apex with involvement of the dural margin of the left cavernous
sinus, Meckel’s cave, and foramen ovale (Figure 5A). Based on clinical features and
neuroimaging results, an extensive evaluation for sarcoidosis and other granulomatous
disorders, IgG4 related disorders, lymphoma or an atypical infectious meningitis was
performed. CSF analysis was unremarkable with 7 leukocytes/mm3, protein 48 mg/dL, and
glucose 54 mg/dL. CSF flow cytometry was negative. An FDG-PET scan with CT showed
non-metabolically active calcific and non-calcific lymph nodes in the mediastinum and
bilateral hilar consistent with sequelae of prior granulomatous disease. The patient
continued with recurrent headaches and ophthalmoplegia despite treatment with
dexamethasone. An attempt to discontinue steroid treatment resulted in exacerbation of
symptoms. Because of progressive brain changes on MRI and persistence of symptoms
despite steroid treatment, the patient underwent a biopsies of the skull base mass
including specimens for NGS. Neuropathological studies of the dural based lesion showed
chronic inflammation and fibrosis (Figure 5C). The specimens contained a moderately
dense infiltrate of lymphocytes, macrophages, and a few plasma cells. Special histological
stains and microbiological studies for bacteria, fungi and mycobacteria were negative.
NGS studies of the dura biopsy showed Delftia acidovorans (or possibly Chryseobacterium
taeanense, which is very closely related to Delftia) and Corynebacterium kroppenstedtii.
Although Delftia was seen in other samples, the relative proportion of Delftia reads in this
patient was much higher (45% of non-human and non-contaminant reads, see
Supplementary Table 2) than in any other patient. Because NGS findings were not validated
by other microbiological or morphological approaches, these results were considered
indeterminate. The patient received then treatment consisting of combination IV antibiotic
therapy with ceftriaxone plus oral moxifloxacin. The dexamethasone dose was tapered and
later discontinued. The patient reported improvement in his headache but persistant
diplopia. A brain and orbital MRI performed at the end of the antibiotic treatment and 4
months later showed decreased meningeal and dural enhancement along the anteromedial
left temporal lobe margin and tentorial leaflet as well as optic nerve dural sheath (Figure
5B). Four months post-treatment the patient reported no retro-orbital headaches or
sensory abnormalities on his face, but persistence of horizontal diplopia. There was no
ptosis but there was a residual palsy of the left VI cranial nerve. Although the NGS findings
were indeterminate, the patient response to antibiotics suggests he experienced a chronic
process leading to pachymeningeal inflammation possibly triggered by an infection.
Patient PT-7: A patient with a history of Fanconi’s anemia and neurodevelopmental
disorder with new onset weakness and a brain mass.
This 19-year-old male had a previous history of dysgenesis of the corpus callosum and
Fanconi’s anemia treated with cord blood transplant and later marrow stem cell transplant.
He had previous therapeutic whole body radiation at age 3. He had been previously
healthy and without any preceding illness or immunosuppression when he developed new-
onset right-sided weakness and seizures. A brain MRI demonstrated a left-sided brain mass
suspected to be a lymphoma or brain tumor (Figure e-1). A brain biopsy suggested a
diagnosis of possible lymphoma and he was treated initially with IV and intrathecal
dexamethasone via an Ommaya reservoir. The patient deteriorated clinically and was
transferred to our institution for further studies. A re-analysis of the brain biopsy was
inconclusive for diagnosis of lymphoma and a second brain biopsy of the brain mass was
obtained and processed for conventional neuropathological studies. The biopsy showed
mild perivascular chronic inflammation (Figure e-1) comprised mostly of CD3-positive T
cells, rare CD20-positive cells, and acute coagulative necrosis. No granulomas were
identified and multiple stains for mycobacteria, spirochetes and other bacteria and fungal
organisms were negative. Similarly, bacterial and mycobacterial cultures from biopsy
tissue were negative. NGS studies were performed in a portion of fresh frozen brain tissue.
NGS studies did not reveal a clear candidate, but did show an unusually high presence of
Lactococcus lactis, a common additive in dairy products that rarely causes human
infections. 244 reads mapped to the genus Lactococcus, 201 of which were specific to
Lactococcus lactis cremoris. Because of clinical concerns that this patient had chronic
cerebritis, he underwent treatment with Ceftriaxone given concerns that his lesion may
have been triggered by this bacterial infection and that he was at increased risk of infection
based on his previous history of immunosuppression. However, one year later (March
2016), re-analysis of this patient’s data revealed 429 reads from Elizabethkingia, a newly
emerging pathogen that caused significant morbidity in a cluster of cases in Wisconsin. The
genomes for this species were unavailable at the time of the original analysis, but 3
genomes are now available: E. sp. BM10, E. anophelis NUHP1, and E. meningoseptica FMS-
007. The greatest number of matches in this sample was to E. sp. BM10. We also note that
re-analysis of the other 9 patients in this study found no evidence of Elizabethkingia in any
other samples. Followup studies for PT-7 are under way at the time of publication.
Cases with non-specific or negative findings that were clinically useful (Cases PT-1,
PT-3, PT-4, PT-6, and PT-9).
Sequencing yielded no specific findings to support a diagnosis of infection in 5 cases;
however the sequencing results did help to rule out concerns about an active infection and
define more specific treatment approaches in 3 cases. In case PT-3, a 23-year-old woman
who developed status epilepticus following a febrile illness was evaluated extensively
without ascertaining an etiological diagnosis. After all serological and CSF studies were
exhausted, a brain biopsy was performed as there were concerns that a viral CNS infection
had triggered her status epilepticus. NGS studies were negative for viruses, and although
some bacterial species were found these were either ruled out as etiopathogenic agents or
considered contaminants. The patient was finally diagnosed with febrile infection related
epilepsy syndrome (FIRES), a rare epileptic syndrome in young adults of uncertain etiology
[7]. Unfortunately the patient died; post-mortem neuropathological studies showed
marked bilateral hippocampal sclerosis and areas of cortical gliosis in selected regions such
as the insular, temporal and frontal cortices. No hallmarks of viral, bacterial or fungal
infections were noted in histopathological studies. In case PT-4, a 37-year-old man with
leptomeningeal and parenchymal inflammatory disease was suspected to have either
infection, sarcoidosis or CNS lymphoma. NGS showed no evidence of specific infection, a
finding that helped to decide treatment. Based on the results of biopsy and NGS, the patient
was treated aggressively with steroids, and this therapy was associated with improvement
of the neuroinflammation suspected to be associated with a granulomatous disease or
sarcoidosis. Similarly, for case PT-9, a 39 year old woman with a history of headache and a
large right hemisphere intra-parenchymal mass demonstrating non-caseating
granulomatous inflammation on pathology, NGS studies showed no evidence of specific
bacteria or mycobacteria to support a diagnosis of infection. The patient was
recommended for treatment with high dose of steroids with a presumptive diagnosis of
non-infectious granulomatous disease, cerebritis., or possibly isolated neurosarcoidosis.
The patient experienced marked improvement of her symptoms and MRI lesion 3 months
after treatment.
In two cases, neuropathological studies demonstrated that the disease process was
associated with primary tumors of the CNS. In case PT-1, a 32-year-old patient with a
history of psoriatic arthritis previously treated with TNF-alpha inhibitors developed a
myelopathic syndrome. Six months later he was found to have meningitis and a large mass
in the thoracic spinal cord. Although initial CSF studies raised concerns about an infection
due to marked pleocytosis, a spinal cord biopsy later documented the presence of a rare
tumor, a spinal cord glioblastoma that produced a rapid progression of myelopathy and
neoplastic meningitis. NGS studies showed several species of bacteria (Supplementary
Table 3) all of which were suspected to be contaminants. In case PT-6, the initial clinical
profile of the patient had also raised concerns about an infectious process based on
epidemiologic information and rapid progression of the neurological symptoms. A biopsy
of the mass showed a rapidly progressive astrocytoma. DNA sequencing identified 2,854
reads as bacterial and 17 as viral. While the bacterial reads did not reveal a potential
pathogen, 15 of the 17 viral reads mapped to JC polyomavirus, a finding considered to be
incidental, although JC polyomavirus has been implicated in the pathogenesis of
astrocytomas in non-human primates [8], and it may infect astrocytes in humans [9]. Thus
although astrocytoma was detected in the patient, JC polyomavirus may have played a role
in its etiology. Furthermore, even though JC polyomavirus is very common in the general
population, infecting 70 to 90% of humans [10], we did not detect it in any other sample
apart from PT-5 (Supplementary Table 4).
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Supplementary Figure e-1. A patient with history of Fanconi’s anemia and brain malformation with a left hemispheric massA) MR imaging study previous to biopsy which demonstrates a left frontal lobe gadolinium–enhancing lesion. B) MR imaging 12 weeks after diagnosis of cerebritis and antibiotic treatment. C) Focal area of inflammation and perivascular inflammation seen in the brain biopsy (HE&PAS stain). NGS studies demonstrated presence of L. lactis cremoris, a bacteria suspected to be the causative pathogen of the cerebritis.