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.