SUPPLEMENTARY INFORMATION (includes supplementary …



SUPPLEMENTARY INFORMATION (includes supplementary methods, clinical summaries, and supplementary Figure e-1)Supplementary MethodsTissue 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 putational 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 ADDIN EN.CITE <EndNote><Cite ExcludeYear="1"><Author>Morgulis</Author><Year>2006</Year><RecNum>6704</RecNum><DisplayText>[11]</DisplayText><record><rec-number>6704</rec-number><foreign-keys><key app="EN" db-id="fzffee0d8fw0xmeet9655992wpfxp9w2rtzf" timestamp="1442358095">6704</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Morgulis, A.</author><author>Gertz, E. M.</author><author>Schaffer, A. A.</author><author>Agarwala, R.</author></authors></contributors><auth-address>National Center for Biotechnology Information, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20894 USA.</auth-address><titles><title>A fast and symmetric DUST implementation to mask low-complexity DNA sequences</title><secondary-title>J Comput Biol</secondary-title></titles><periodical><full-title>J Comput Biol</full-title></periodical><pages>1028-40</pages><volume>13</volume><number>5</number><keywords><keyword>Genome, Human/*genetics</keyword><keyword>Humans</keyword><keyword>*Pattern Recognition, Automated</keyword><keyword>*Sequence Alignment</keyword><keyword>*Sequence Analysis, DNA</keyword><keyword>*Software</keyword></keywords><dates><year>2006</year><pub-dates><date>Jun</date></pub-dates></dates><isbn>1066-5277 (Print)&#xD;1066-5277 (Linking)</isbn><accession-num>16796549</accession-num><urls><related-urls><url>;[11]. In order to identify common vector contaminants, the database also includes the genome of phiX174, and the UniVec () and EMVec () 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 ADDIN EN.CITE <EndNote><Cite ExcludeYear="1"><Author>Salter</Author><Year>2014</Year><RecNum>3879</RecNum><DisplayText>[12]</DisplayText><record><rec-number>3879</rec-number><foreign-keys><key app="EN" db-id="fzffee0d8fw0xmeet9655992wpfxp9w2rtzf" timestamp="1418101952">3879</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Salter, S. J.</author><author>Cox, M. J.</author><author>Turek, E. M.</author><author>Calus, S. T.</author><author>Cookson, W. O.</author><author>Moffatt, M. F.</author><author>Turner, P.</author><author>Parkhill, J.</author><author>Loman, N. J.</author><author>Walker, A. W.</author></authors></contributors><auth-address>Pathogen Genomics Group, Wellcome Trust Sanger Institute, Hinxton, UK. sb18@sanger.ac.uk.</auth-address><titles><title>Reagent and laboratory contamination can critically impact sequence-based microbiome analyses</title><secondary-title>BMC Biol</secondary-title><alt-title>BMC biology</alt-title></titles><periodical><full-title>BMC Biol</full-title><abbr-1>BMC biology</abbr-1></periodical><alt-periodical><full-title>BMC Biol</full-title><abbr-1>BMC biology</abbr-1></alt-periodical><pages>87</pages><volume>12</volume><number>1</number><edition>2014/11/13</edition><dates><year>2014</year></dates><isbn>1741-7007</isbn><accession-num>25387460</accession-num><urls></urls><custom2>Pmc4228153</custom2><electronic-resource-num>10.1186/s12915-014-0087-z</electronic-resource-num><remote-database-provider>NLM</remote-database-provider><language>eng</language></record></Cite></EndNote>[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 ADDIN EN.CITE <EndNote><Cite ExcludeYear="1"><Author>Segata</Author><Year>2012</Year><RecNum>3891</RecNum><DisplayText>[13]</DisplayText><record><rec-number>3891</rec-number><foreign-keys><key app="EN" db-id="fzffee0d8fw0xmeet9655992wpfxp9w2rtzf" timestamp="1418101953">3891</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Segata, N.</author><author>Waldron, L.</author><author>Ballarini, A.</author><author>Narasimhan, V.</author><author>Jousson, O.</author><author>Huttenhower, C.</author></authors></contributors><auth-address>Department of Biostatistics, Harvard School of Public Health, Boston, Massachusetts, USA.</auth-address><titles><title>Metagenomic microbial community profiling using unique clade-specific marker genes</title><secondary-title>Nat Methods</secondary-title><alt-title>Nature methods</alt-title></titles><periodical><full-title>Nat Methods</full-title><abbr-1>Nature methods</abbr-1></periodical><alt-periodical><full-title>Nat Methods</full-title><abbr-1>Nature methods</abbr-1></alt-periodical><pages>811-4</pages><volume>9</volume><number>8</number><edition>2012/06/13</edition><keywords><keyword>Bacteria/*classification/*genetics/isolation &amp; purification</keyword><keyword>Genetic Markers/*genetics</keyword><keyword>Humans</keyword><keyword>*Metagenomics</keyword><keyword>*Phylogeny</keyword><keyword>Reproducibility of Results</keyword></keywords><dates><year>2012</year><pub-dates><date>Aug</date></pub-dates></dates><isbn>1548-7091</isbn><accession-num>22688413</accession-num><urls></urls><custom2>Pmc3443552</custom2><custom6>Nihms397911</custom6><electronic-resource-num>10.1038/nmeth.2066</electronic-resource-num><remote-database-provider>NLM</remote-database-provider><language>eng</language></record></Cite></EndNote>[13] and DIAMOND ADDIN EN.CITE <EndNote><Cite ExcludeYear="1"><Author>Buchfink</Author><Year>2015</Year><RecNum>6705</RecNum><DisplayText>[14]</DisplayText><record><rec-number>6705</rec-number><foreign-keys><key app="EN" db-id="fzffee0d8fw0xmeet9655992wpfxp9w2rtzf" timestamp="1442358183">6705</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Buchfink, B.</author><author>Xie, C.</author><author>Huson, D. H.</author></authors></contributors><auth-address>Department of Computer Science and Center for Bioinformatics, University of Tubingen, Tubingen, Germany.&#xD;1] Singapore Centre on Environmental Life Sciences Engineering, School of Biological Sciences, Nanyang Technological University, Singapore. [2] Life Sciences Institute, National University of Singapore, Singapore.&#xD;1] Department of Computer Science and Center for Bioinformatics, University of Tubingen, Tubingen, Germany. [2] Singapore Centre on Environmental Life Sciences Engineering, School of Biological Sciences, Nanyang Technological University, Singapore.</auth-address><titles><title>Fast and sensitive protein alignment using DIAMOND</title><secondary-title>Nat Methods</secondary-title></titles><periodical><full-title>Nat Methods</full-title><abbr-1>Nature methods</abbr-1></periodical><pages>59-60</pages><volume>12</volume><number>1</number><keywords><keyword>Algorithms</keyword><keyword>Base Sequence</keyword><keyword>Humans</keyword><keyword>Metagenomics/*methods</keyword><keyword>Microbiota/genetics</keyword><keyword>Sensitivity and Specificity</keyword><keyword>Sequence Alignment/*methods</keyword><keyword>Sequence Analysis, DNA</keyword><keyword>*Software</keyword></keywords><dates><year>2015</year><pub-dates><date>Jan</date></pub-dates></dates><isbn>1548-7105 (Electronic)&#xD;1548-7091 (Linking)</isbn><accession-num>25402007</accession-num><urls><related-urls><url>;[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 SUMMARIESCases with a high degree of diagnostic confidence and positive pathogen identificationPatient 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 PEVuZE5vdGU+PENpdGUgRXhjbHVkZVllYXI9IjEiPjxBdXRob3I+Qm9laG1lPC9BdXRob3I+PFll

YXI+MjAxMDwvWWVhcj48UmVjTnVtPjY3MDM8L1JlY051bT48RGlzcGxheVRleHQ+WzFdPC9EaXNw

bGF5VGV4dD48cmVjb3JkPjxyZWMtbnVtYmVyPjY3MDM8L3JlYy1udW1iZXI+PGZvcmVpZ24ta2V5

cz48a2V5IGFwcD0iRU4iIGRiLWlkPSJmemZmZWUwZDhmdzB4bWVldDk2NTU5OTJ3cGZ4cDl3MnJ0

emYiIHRpbWVzdGFtcD0iMTQ0MjI5NDkzMiI+NjcwMzwva2V5PjwvZm9yZWlnbi1rZXlzPjxyZWYt

dHlwZSBuYW1lPSJKb3VybmFsIEFydGljbGUiPjE3PC9yZWYtdHlwZT48Y29udHJpYnV0b3JzPjxh

dXRob3JzPjxhdXRob3I+Qm9laG1lLCBDLiBDLjwvYXV0aG9yPjxhdXRob3I+TmFiZXRhLCBQLjwv

YXV0aG9yPjxhdXRob3I+SGlsbGVtYW5uLCBELjwvYXV0aG9yPjxhdXRob3I+Tmljb2wsIE0uIFAu

PC9hdXRob3I+PGF1dGhvcj5TaGVuYWksIFMuPC9hdXRob3I+PGF1dGhvcj5LcmFwcCwgRi48L2F1

dGhvcj48YXV0aG9yPkFsbGVuLCBKLjwvYXV0aG9yPjxhdXRob3I+VGFoaXJsaSwgUi48L2F1dGhv

cj48YXV0aG9yPkJsYWtlbW9yZSwgUi48L2F1dGhvcj48YXV0aG9yPlJ1c3RvbWplZSwgUi48L2F1

dGhvcj48YXV0aG9yPk1pbG92aWMsIEEuPC9hdXRob3I+PGF1dGhvcj5Kb25lcywgTS48L2F1dGhv

cj48YXV0aG9yPk8mYXBvcztCcmllbiwgUy4gTS48L2F1dGhvcj48YXV0aG9yPlBlcnNpbmcsIEQu

IEguPC9hdXRob3I+PGF1dGhvcj5SdWVzY2gtR2VyZGVzLCBTLjwvYXV0aG9yPjxhdXRob3I+R290

dXp6bywgRS48L2F1dGhvcj48YXV0aG9yPlJvZHJpZ3VlcywgQy48L2F1dGhvcj48YXV0aG9yPkFs

bGFuZCwgRC48L2F1dGhvcj48YXV0aG9yPlBlcmtpbnMsIE0uIEQuPC9hdXRob3I+PC9hdXRob3Jz

PjwvY29udHJpYnV0b3JzPjxhdXRoLWFkZHJlc3M+Rm91bmRhdGlvbiBmb3IgSW5ub3ZhdGl2ZSBO

ZXcgRGlhZ25vc3RpY3MsIEdlbmV2YSwgU3dpdHplcmxhbmQuIGNhdGhhcmluYS5ib2VobWVAZmlu

ZGRpYWdub3N0aWNzLm9yZzwvYXV0aC1hZGRyZXNzPjx0aXRsZXM+PHRpdGxlPlJhcGlkIG1vbGVj

dWxhciBkZXRlY3Rpb24gb2YgdHViZXJjdWxvc2lzIGFuZCByaWZhbXBpbiByZXNpc3RhbmNlPC90

aXRsZT48c2Vjb25kYXJ5LXRpdGxlPk4gRW5nbCBKIE1lZDwvc2Vjb25kYXJ5LXRpdGxlPjxhbHQt

dGl0bGU+VGhlIE5ldyBFbmdsYW5kIGpvdXJuYWwgb2YgbWVkaWNpbmU8L2FsdC10aXRsZT48L3Rp

dGxlcz48cGVyaW9kaWNhbD48ZnVsbC10aXRsZT5OIEVuZ2wgSiBNZWQ8L2Z1bGwtdGl0bGU+PGFi

YnItMT5UaGUgTmV3IEVuZ2xhbmQgam91cm5hbCBvZiBtZWRpY2luZTwvYWJici0xPjwvcGVyaW9k

aWNhbD48YWx0LXBlcmlvZGljYWw+PGZ1bGwtdGl0bGU+TiBFbmdsIEogTWVkPC9mdWxsLXRpdGxl

PjxhYmJyLTE+VGhlIE5ldyBFbmdsYW5kIGpvdXJuYWwgb2YgbWVkaWNpbmU8L2FiYnItMT48L2Fs

dC1wZXJpb2RpY2FsPjxwYWdlcz4xMDA1LTE1PC9wYWdlcz48dm9sdW1lPjM2Mzwvdm9sdW1lPjxu

dW1iZXI+MTE8L251bWJlcj48ZWRpdGlvbj4yMDEwLzA5LzEwPC9lZGl0aW9uPjxrZXl3b3Jkcz48

a2V5d29yZD5BZG9sZXNjZW50PC9rZXl3b3JkPjxrZXl3b3JkPkFkdWx0PC9rZXl3b3JkPjxrZXl3

b3JkPkFnZWQ8L2tleXdvcmQ+PGtleXdvcmQ+QWdlZCwgODAgYW5kIG92ZXI8L2tleXdvcmQ+PGtl

eXdvcmQ+QW50aXR1YmVyY3VsYXIgQWdlbnRzLypwaGFybWFjb2xvZ3k8L2tleXdvcmQ+PGtleXdv

cmQ+QmFjdGVyaWFsIFByb3RlaW5zL2dlbmV0aWNzPC9rZXl3b3JkPjxrZXl3b3JkPipEcnVnIFJl

c2lzdGFuY2UsIEJhY3RlcmlhbDwva2V5d29yZD48a2V5d29yZD5GZW1hbGU8L2tleXdvcmQ+PGtl

eXdvcmQ+SHVtYW5zPC9rZXl3b3JkPjxrZXl3b3JkPk1hbGU8L2tleXdvcmQ+PGtleXdvcmQ+TWlj

cm9iaWFsIFNlbnNpdGl2aXR5IFRlc3RzL21ldGhvZHM8L2tleXdvcmQ+PGtleXdvcmQ+TWlkZGxl

IEFnZWQ8L2tleXdvcmQ+PGtleXdvcmQ+TXljb2JhY3Rlcml1bSB0dWJlcmN1bG9zaXMvKmRydWcg

ZWZmZWN0cy9nZW5ldGljczwva2V5d29yZD48a2V5d29yZD5Qb2x5bWVyYXNlIENoYWluIFJlYWN0

aW9uL2luc3RydW1lbnRhdGlvbi8qbWV0aG9kczwva2V5d29yZD48a2V5d29yZD5Qcm9zcGVjdGl2

ZSBTdHVkaWVzPC9rZXl3b3JkPjxrZXl3b3JkPlJlZmVyZW5jZSBTdGFuZGFyZHM8L2tleXdvcmQ+

PGtleXdvcmQ+UmlmYW1waW4vKnBoYXJtYWNvbG9neTwva2V5d29yZD48a2V5d29yZD5TZW5zaXRp

dml0eSBhbmQgU3BlY2lmaWNpdHk8L2tleXdvcmQ+PGtleXdvcmQ+U3B1dHVtL21pY3JvYmlvbG9n

eTwva2V5d29yZD48a2V5d29yZD5UdWJlcmN1bG9zaXMvZGlhZ25vc2lzLyptaWNyb2Jpb2xvZ3k8

L2tleXdvcmQ+PGtleXdvcmQ+VHViZXJjdWxvc2lzLCBNdWx0aWRydWctUmVzaXN0YW50LypkaWFn

bm9zaXMvbWljcm9iaW9sb2d5PC9rZXl3b3JkPjxrZXl3b3JkPllvdW5nIEFkdWx0PC9rZXl3b3Jk

Pjwva2V5d29yZHM+PGRhdGVzPjx5ZWFyPjIwMTA8L3llYXI+PHB1Yi1kYXRlcz48ZGF0ZT5TZXAg

OTwvZGF0ZT48L3B1Yi1kYXRlcz48L2RhdGVzPjxpc2JuPjAwMjgtNDc5MzwvaXNibj48YWNjZXNz

aW9uLW51bT4yMDgyNTMxMzwvYWNjZXNzaW9uLW51bT48dXJscz48L3VybHM+PGN1c3RvbTI+UG1j

Mjk0Nzc5OTwvY3VzdG9tMj48Y3VzdG9tNj5OaWhtczIzNTUxNzwvY3VzdG9tNj48ZWxlY3Ryb25p

Yy1yZXNvdXJjZS1udW0+MTAuMTA1Ni9ORUpNb2EwOTA3ODQ3PC9lbGVjdHJvbmljLXJlc291cmNl

LW51bT48cmVtb3RlLWRhdGFiYXNlLXByb3ZpZGVyPk5MTTwvcmVtb3RlLWRhdGFiYXNlLXByb3Zp

ZGVyPjxsYW5ndWFnZT5lbmc8L2xhbmd1YWdlPjwvcmVjb3JkPjwvQ2l0ZT48L0VuZE5vdGU+

ADDIN EN.CITE PEVuZE5vdGU+PENpdGUgRXhjbHVkZVllYXI9IjEiPjxBdXRob3I+Qm9laG1lPC9BdXRob3I+PFll

YXI+MjAxMDwvWWVhcj48UmVjTnVtPjY3MDM8L1JlY051bT48RGlzcGxheVRleHQ+WzFdPC9EaXNw

bGF5VGV4dD48cmVjb3JkPjxyZWMtbnVtYmVyPjY3MDM8L3JlYy1udW1iZXI+PGZvcmVpZ24ta2V5

cz48a2V5IGFwcD0iRU4iIGRiLWlkPSJmemZmZWUwZDhmdzB4bWVldDk2NTU5OTJ3cGZ4cDl3MnJ0

emYiIHRpbWVzdGFtcD0iMTQ0MjI5NDkzMiI+NjcwMzwva2V5PjwvZm9yZWlnbi1rZXlzPjxyZWYt

dHlwZSBuYW1lPSJKb3VybmFsIEFydGljbGUiPjE3PC9yZWYtdHlwZT48Y29udHJpYnV0b3JzPjxh

dXRob3JzPjxhdXRob3I+Qm9laG1lLCBDLiBDLjwvYXV0aG9yPjxhdXRob3I+TmFiZXRhLCBQLjwv

YXV0aG9yPjxhdXRob3I+SGlsbGVtYW5uLCBELjwvYXV0aG9yPjxhdXRob3I+Tmljb2wsIE0uIFAu

PC9hdXRob3I+PGF1dGhvcj5TaGVuYWksIFMuPC9hdXRob3I+PGF1dGhvcj5LcmFwcCwgRi48L2F1

dGhvcj48YXV0aG9yPkFsbGVuLCBKLjwvYXV0aG9yPjxhdXRob3I+VGFoaXJsaSwgUi48L2F1dGhv

cj48YXV0aG9yPkJsYWtlbW9yZSwgUi48L2F1dGhvcj48YXV0aG9yPlJ1c3RvbWplZSwgUi48L2F1

dGhvcj48YXV0aG9yPk1pbG92aWMsIEEuPC9hdXRob3I+PGF1dGhvcj5Kb25lcywgTS48L2F1dGhv

cj48YXV0aG9yPk8mYXBvcztCcmllbiwgUy4gTS48L2F1dGhvcj48YXV0aG9yPlBlcnNpbmcsIEQu

IEguPC9hdXRob3I+PGF1dGhvcj5SdWVzY2gtR2VyZGVzLCBTLjwvYXV0aG9yPjxhdXRob3I+R290

dXp6bywgRS48L2F1dGhvcj48YXV0aG9yPlJvZHJpZ3VlcywgQy48L2F1dGhvcj48YXV0aG9yPkFs

bGFuZCwgRC48L2F1dGhvcj48YXV0aG9yPlBlcmtpbnMsIE0uIEQuPC9hdXRob3I+PC9hdXRob3Jz

PjwvY29udHJpYnV0b3JzPjxhdXRoLWFkZHJlc3M+Rm91bmRhdGlvbiBmb3IgSW5ub3ZhdGl2ZSBO

ZXcgRGlhZ25vc3RpY3MsIEdlbmV2YSwgU3dpdHplcmxhbmQuIGNhdGhhcmluYS5ib2VobWVAZmlu

ZGRpYWdub3N0aWNzLm9yZzwvYXV0aC1hZGRyZXNzPjx0aXRsZXM+PHRpdGxlPlJhcGlkIG1vbGVj

dWxhciBkZXRlY3Rpb24gb2YgdHViZXJjdWxvc2lzIGFuZCByaWZhbXBpbiByZXNpc3RhbmNlPC90

aXRsZT48c2Vjb25kYXJ5LXRpdGxlPk4gRW5nbCBKIE1lZDwvc2Vjb25kYXJ5LXRpdGxlPjxhbHQt

dGl0bGU+VGhlIE5ldyBFbmdsYW5kIGpvdXJuYWwgb2YgbWVkaWNpbmU8L2FsdC10aXRsZT48L3Rp

dGxlcz48cGVyaW9kaWNhbD48ZnVsbC10aXRsZT5OIEVuZ2wgSiBNZWQ8L2Z1bGwtdGl0bGU+PGFi

YnItMT5UaGUgTmV3IEVuZ2xhbmQgam91cm5hbCBvZiBtZWRpY2luZTwvYWJici0xPjwvcGVyaW9k

aWNhbD48YWx0LXBlcmlvZGljYWw+PGZ1bGwtdGl0bGU+TiBFbmdsIEogTWVkPC9mdWxsLXRpdGxl

PjxhYmJyLTE+VGhlIE5ldyBFbmdsYW5kIGpvdXJuYWwgb2YgbWVkaWNpbmU8L2FiYnItMT48L2Fs

dC1wZXJpb2RpY2FsPjxwYWdlcz4xMDA1LTE1PC9wYWdlcz48dm9sdW1lPjM2Mzwvdm9sdW1lPjxu

dW1iZXI+MTE8L251bWJlcj48ZWRpdGlvbj4yMDEwLzA5LzEwPC9lZGl0aW9uPjxrZXl3b3Jkcz48

a2V5d29yZD5BZG9sZXNjZW50PC9rZXl3b3JkPjxrZXl3b3JkPkFkdWx0PC9rZXl3b3JkPjxrZXl3

b3JkPkFnZWQ8L2tleXdvcmQ+PGtleXdvcmQ+QWdlZCwgODAgYW5kIG92ZXI8L2tleXdvcmQ+PGtl

eXdvcmQ+QW50aXR1YmVyY3VsYXIgQWdlbnRzLypwaGFybWFjb2xvZ3k8L2tleXdvcmQ+PGtleXdv

cmQ+QmFjdGVyaWFsIFByb3RlaW5zL2dlbmV0aWNzPC9rZXl3b3JkPjxrZXl3b3JkPipEcnVnIFJl

c2lzdGFuY2UsIEJhY3RlcmlhbDwva2V5d29yZD48a2V5d29yZD5GZW1hbGU8L2tleXdvcmQ+PGtl

eXdvcmQ+SHVtYW5zPC9rZXl3b3JkPjxrZXl3b3JkPk1hbGU8L2tleXdvcmQ+PGtleXdvcmQ+TWlj

cm9iaWFsIFNlbnNpdGl2aXR5IFRlc3RzL21ldGhvZHM8L2tleXdvcmQ+PGtleXdvcmQ+TWlkZGxl

IEFnZWQ8L2tleXdvcmQ+PGtleXdvcmQ+TXljb2JhY3Rlcml1bSB0dWJlcmN1bG9zaXMvKmRydWcg

ZWZmZWN0cy9nZW5ldGljczwva2V5d29yZD48a2V5d29yZD5Qb2x5bWVyYXNlIENoYWluIFJlYWN0

aW9uL2luc3RydW1lbnRhdGlvbi8qbWV0aG9kczwva2V5d29yZD48a2V5d29yZD5Qcm9zcGVjdGl2

ZSBTdHVkaWVzPC9rZXl3b3JkPjxrZXl3b3JkPlJlZmVyZW5jZSBTdGFuZGFyZHM8L2tleXdvcmQ+

PGtleXdvcmQ+UmlmYW1waW4vKnBoYXJtYWNvbG9neTwva2V5d29yZD48a2V5d29yZD5TZW5zaXRp

dml0eSBhbmQgU3BlY2lmaWNpdHk8L2tleXdvcmQ+PGtleXdvcmQ+U3B1dHVtL21pY3JvYmlvbG9n

eTwva2V5d29yZD48a2V5d29yZD5UdWJlcmN1bG9zaXMvZGlhZ25vc2lzLyptaWNyb2Jpb2xvZ3k8

L2tleXdvcmQ+PGtleXdvcmQ+VHViZXJjdWxvc2lzLCBNdWx0aWRydWctUmVzaXN0YW50LypkaWFn

bm9zaXMvbWljcm9iaW9sb2d5PC9rZXl3b3JkPjxrZXl3b3JkPllvdW5nIEFkdWx0PC9rZXl3b3Jk

Pjwva2V5d29yZHM+PGRhdGVzPjx5ZWFyPjIwMTA8L3llYXI+PHB1Yi1kYXRlcz48ZGF0ZT5TZXAg

OTwvZGF0ZT48L3B1Yi1kYXRlcz48L2RhdGVzPjxpc2JuPjAwMjgtNDc5MzwvaXNibj48YWNjZXNz

aW9uLW51bT4yMDgyNTMxMzwvYWNjZXNzaW9uLW51bT48dXJscz48L3VybHM+PGN1c3RvbTI+UG1j

Mjk0Nzc5OTwvY3VzdG9tMj48Y3VzdG9tNj5OaWhtczIzNTUxNzwvY3VzdG9tNj48ZWxlY3Ryb25p

Yy1yZXNvdXJjZS1udW0+MTAuMTA1Ni9ORUpNb2EwOTA3ODQ3PC9lbGVjdHJvbmljLXJlc291cmNl

LW51bT48cmVtb3RlLWRhdGFiYXNlLXByb3ZpZGVyPk5MTTwvcmVtb3RlLWRhdGFiYXNlLXByb3Zp

ZGVyPjxsYW5ndWFnZT5lbmc8L2xhbmd1YWdlPjwvcmVjb3JkPjwvQ2l0ZT48L0VuZE5vdGU+

ADDIN EN.CITE.DATA [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 PEVuZE5vdGU+PENpdGUgRXhjbHVkZVllYXI9IjEiPjxBdXRob3I+U2ltbmVyPC9BdXRob3I+PFll

YXI+MjAxMzwvWWVhcj48UmVjTnVtPjY3MzE8L1JlY051bT48RGlzcGxheVRleHQ+WzJdPC9EaXNw

bGF5VGV4dD48cmVjb3JkPjxyZWMtbnVtYmVyPjY3MzE8L3JlYy1udW1iZXI+PGZvcmVpZ24ta2V5

cz48a2V5IGFwcD0iRU4iIGRiLWlkPSJmemZmZWUwZDhmdzB4bWVldDk2NTU5OTJ3cGZ4cDl3MnJ0

emYiIHRpbWVzdGFtcD0iMTQ0MjM2MDU0OSI+NjczMTwva2V5PjwvZm9yZWlnbi1rZXlzPjxyZWYt

dHlwZSBuYW1lPSJKb3VybmFsIEFydGljbGUiPjE3PC9yZWYtdHlwZT48Y29udHJpYnV0b3JzPjxh

dXRob3JzPjxhdXRob3I+U2ltbmVyLCBQLiBKLjwvYXV0aG9yPjxhdXRob3I+VWhsLCBKLiBSLjwv

YXV0aG9yPjxhdXRob3I+SGFsbCwgTC48L2F1dGhvcj48YXV0aG9yPldlYmVyLCBNLiBNLjwvYXV0

aG9yPjxhdXRob3I+V2FsY2hhaywgUi4gQy48L2F1dGhvcj48YXV0aG9yPkJ1Y2t3YWx0ZXIsIFMu

PC9hdXRob3I+PGF1dGhvcj5XZW5nZW5hY2ssIE4uIEwuPC9hdXRob3I+PC9hdXRob3JzPjwvY29u

dHJpYnV0b3JzPjxhdXRoLWFkZHJlc3M+RGl2aXNpb24gb2YgQ2xpbmljYWwgTWljcm9iaW9sb2d5

LCBEZXBhcnRtZW50IG9mIExhYm9yYXRvcnkgTWVkaWNpbmUgYW5kIFBhdGhvbG9neSwgTWF5byBD

bGluaWMsIFJvY2hlc3RlciwgTWlubmVzb3RhLCBVU0EuPC9hdXRoLWFkZHJlc3M+PHRpdGxlcz48

dGl0bGU+QnJvYWQtcmFuZ2UgZGlyZWN0IGRldGVjdGlvbiBhbmQgaWRlbnRpZmljYXRpb24gb2Yg

ZnVuZ2kgYnkgdXNlIG9mIHRoZSBQTEVYLUlEIFBDUi1lbGVjdHJvc3ByYXkgaW9uaXphdGlvbiBt

YXNzIHNwZWN0cm9tZXRyeSAoRVNJLU1TKSBzeXN0ZW08L3RpdGxlPjxzZWNvbmRhcnktdGl0bGU+

SiBDbGluIE1pY3JvYmlvbDwvc2Vjb25kYXJ5LXRpdGxlPjwvdGl0bGVzPjxwZXJpb2RpY2FsPjxm

dWxsLXRpdGxlPkogQ2xpbiBNaWNyb2Jpb2w8L2Z1bGwtdGl0bGU+PGFiYnItMT5Kb3VybmFsIG9m

IGNsaW5pY2FsIG1pY3JvYmlvbG9neTwvYWJici0xPjwvcGVyaW9kaWNhbD48cGFnZXM+MTY5OS03

MDY8L3BhZ2VzPjx2b2x1bWU+NTE8L3ZvbHVtZT48bnVtYmVyPjY8L251bWJlcj48a2V5d29yZHM+

PGtleXdvcmQ+RnVuZ2kvKmNoZW1pc3RyeS9jbGFzc2lmaWNhdGlvbi8qaXNvbGF0aW9uICZhbXA7

IHB1cmlmaWNhdGlvbjwva2V5d29yZD48a2V5d29yZD5IdW1hbnM8L2tleXdvcmQ+PGtleXdvcmQ+

TWljcm9iaW9sb2dpY2FsIFRlY2huaXF1ZXMvKm1ldGhvZHM8L2tleXdvcmQ+PGtleXdvcmQ+TW9s

ZWN1bGFyIERpYWdub3N0aWMgVGVjaG5pcXVlcy8qbWV0aG9kczwva2V5d29yZD48a2V5d29yZD5N

eWNvbG9neS8qbWV0aG9kczwva2V5d29yZD48a2V5d29yZD5NeWNvc2VzL2RpYWdub3Npczwva2V5

d29yZD48a2V5d29yZD5Qb2x5bWVyYXNlIENoYWluIFJlYWN0aW9uLyptZXRob2RzPC9rZXl3b3Jk

PjxrZXl3b3JkPlJlc3BpcmF0b3J5IFRyYWN0IEluZmVjdGlvbnMvbWljcm9iaW9sb2d5PC9rZXl3

b3JkPjxrZXl3b3JkPlNlbnNpdGl2aXR5IGFuZCBTcGVjaWZpY2l0eTwva2V5d29yZD48a2V5d29y

ZD5TcGVjdHJvbWV0cnksIE1hc3MsIEVsZWN0cm9zcHJheSBJb25pemF0aW9uLyptZXRob2RzPC9r

ZXl3b3JkPjwva2V5d29yZHM+PGRhdGVzPjx5ZWFyPjIwMTM8L3llYXI+PHB1Yi1kYXRlcz48ZGF0

ZT5KdW48L2RhdGU+PC9wdWItZGF0ZXM+PC9kYXRlcz48aXNibj4xMDk4LTY2MFggKEVsZWN0cm9u

aWMpJiN4RDswMDk1LTExMzcgKExpbmtpbmcpPC9pc2JuPjxhY2Nlc3Npb24tbnVtPjIzNTE1NTQw

PC9hY2Nlc3Npb24tbnVtPjx1cmxzPjxyZWxhdGVkLXVybHM+PHVybD5odHRwOi8vd3d3Lm5jYmku

bmxtLm5paC5nb3YvcHVibWVkLzIzNTE1NTQwPC91cmw+PC9yZWxhdGVkLXVybHM+PC91cmxzPjxj

dXN0b20yPlBNQzM3MTYwODg8L2N1c3RvbTI+PGVsZWN0cm9uaWMtcmVzb3VyY2UtbnVtPjEwLjEx

MjgvSkNNLjAzMjgyLTEyPC9lbGVjdHJvbmljLXJlc291cmNlLW51bT48L3JlY29yZD48L0NpdGU+

PC9FbmROb3RlPn==

ADDIN EN.CITE PEVuZE5vdGU+PENpdGUgRXhjbHVkZVllYXI9IjEiPjxBdXRob3I+U2ltbmVyPC9BdXRob3I+PFll

YXI+MjAxMzwvWWVhcj48UmVjTnVtPjY3MzE8L1JlY051bT48RGlzcGxheVRleHQ+WzJdPC9EaXNw

bGF5VGV4dD48cmVjb3JkPjxyZWMtbnVtYmVyPjY3MzE8L3JlYy1udW1iZXI+PGZvcmVpZ24ta2V5

cz48a2V5IGFwcD0iRU4iIGRiLWlkPSJmemZmZWUwZDhmdzB4bWVldDk2NTU5OTJ3cGZ4cDl3MnJ0

emYiIHRpbWVzdGFtcD0iMTQ0MjM2MDU0OSI+NjczMTwva2V5PjwvZm9yZWlnbi1rZXlzPjxyZWYt

dHlwZSBuYW1lPSJKb3VybmFsIEFydGljbGUiPjE3PC9yZWYtdHlwZT48Y29udHJpYnV0b3JzPjxh

dXRob3JzPjxhdXRob3I+U2ltbmVyLCBQLiBKLjwvYXV0aG9yPjxhdXRob3I+VWhsLCBKLiBSLjwv

YXV0aG9yPjxhdXRob3I+SGFsbCwgTC48L2F1dGhvcj48YXV0aG9yPldlYmVyLCBNLiBNLjwvYXV0

aG9yPjxhdXRob3I+V2FsY2hhaywgUi4gQy48L2F1dGhvcj48YXV0aG9yPkJ1Y2t3YWx0ZXIsIFMu

PC9hdXRob3I+PGF1dGhvcj5XZW5nZW5hY2ssIE4uIEwuPC9hdXRob3I+PC9hdXRob3JzPjwvY29u

dHJpYnV0b3JzPjxhdXRoLWFkZHJlc3M+RGl2aXNpb24gb2YgQ2xpbmljYWwgTWljcm9iaW9sb2d5

LCBEZXBhcnRtZW50IG9mIExhYm9yYXRvcnkgTWVkaWNpbmUgYW5kIFBhdGhvbG9neSwgTWF5byBD

bGluaWMsIFJvY2hlc3RlciwgTWlubmVzb3RhLCBVU0EuPC9hdXRoLWFkZHJlc3M+PHRpdGxlcz48

dGl0bGU+QnJvYWQtcmFuZ2UgZGlyZWN0IGRldGVjdGlvbiBhbmQgaWRlbnRpZmljYXRpb24gb2Yg

ZnVuZ2kgYnkgdXNlIG9mIHRoZSBQTEVYLUlEIFBDUi1lbGVjdHJvc3ByYXkgaW9uaXphdGlvbiBt

YXNzIHNwZWN0cm9tZXRyeSAoRVNJLU1TKSBzeXN0ZW08L3RpdGxlPjxzZWNvbmRhcnktdGl0bGU+

SiBDbGluIE1pY3JvYmlvbDwvc2Vjb25kYXJ5LXRpdGxlPjwvdGl0bGVzPjxwZXJpb2RpY2FsPjxm

dWxsLXRpdGxlPkogQ2xpbiBNaWNyb2Jpb2w8L2Z1bGwtdGl0bGU+PGFiYnItMT5Kb3VybmFsIG9m

IGNsaW5pY2FsIG1pY3JvYmlvbG9neTwvYWJici0xPjwvcGVyaW9kaWNhbD48cGFnZXM+MTY5OS03

MDY8L3BhZ2VzPjx2b2x1bWU+NTE8L3ZvbHVtZT48bnVtYmVyPjY8L251bWJlcj48a2V5d29yZHM+

PGtleXdvcmQ+RnVuZ2kvKmNoZW1pc3RyeS9jbGFzc2lmaWNhdGlvbi8qaXNvbGF0aW9uICZhbXA7

IHB1cmlmaWNhdGlvbjwva2V5d29yZD48a2V5d29yZD5IdW1hbnM8L2tleXdvcmQ+PGtleXdvcmQ+

TWljcm9iaW9sb2dpY2FsIFRlY2huaXF1ZXMvKm1ldGhvZHM8L2tleXdvcmQ+PGtleXdvcmQ+TW9s

ZWN1bGFyIERpYWdub3N0aWMgVGVjaG5pcXVlcy8qbWV0aG9kczwva2V5d29yZD48a2V5d29yZD5N

eWNvbG9neS8qbWV0aG9kczwva2V5d29yZD48a2V5d29yZD5NeWNvc2VzL2RpYWdub3Npczwva2V5

d29yZD48a2V5d29yZD5Qb2x5bWVyYXNlIENoYWluIFJlYWN0aW9uLyptZXRob2RzPC9rZXl3b3Jk

PjxrZXl3b3JkPlJlc3BpcmF0b3J5IFRyYWN0IEluZmVjdGlvbnMvbWljcm9iaW9sb2d5PC9rZXl3

b3JkPjxrZXl3b3JkPlNlbnNpdGl2aXR5IGFuZCBTcGVjaWZpY2l0eTwva2V5d29yZD48a2V5d29y

ZD5TcGVjdHJvbWV0cnksIE1hc3MsIEVsZWN0cm9zcHJheSBJb25pemF0aW9uLyptZXRob2RzPC9r

ZXl3b3JkPjwva2V5d29yZHM+PGRhdGVzPjx5ZWFyPjIwMTM8L3llYXI+PHB1Yi1kYXRlcz48ZGF0

ZT5KdW48L2RhdGU+PC9wdWItZGF0ZXM+PC9kYXRlcz48aXNibj4xMDk4LTY2MFggKEVsZWN0cm9u

aWMpJiN4RDswMDk1LTExMzcgKExpbmtpbmcpPC9pc2JuPjxhY2Nlc3Npb24tbnVtPjIzNTE1NTQw

PC9hY2Nlc3Npb24tbnVtPjx1cmxzPjxyZWxhdGVkLXVybHM+PHVybD5odHRwOi8vd3d3Lm5jYmku

bmxtLm5paC5nb3YvcHVibWVkLzIzNTE1NTQwPC91cmw+PC9yZWxhdGVkLXVybHM+PC91cmxzPjxj

dXN0b20yPlBNQzM3MTYwODg8L2N1c3RvbTI+PGVsZWN0cm9uaWMtcmVzb3VyY2UtbnVtPjEwLjEx

MjgvSkNNLjAzMjgyLTEyPC9lbGVjdHJvbmljLXJlc291cmNlLW51bT48L3JlY29yZD48L0NpdGU+

PC9FbmROb3RlPn==

ADDIN EN.CITE.DATA [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 ADDIN EN.CITE <EndNote><Cite ExcludeYear="1"><Author>Altschul</Author><Year>1997</Year><RecNum>691</RecNum><DisplayText>[3]</DisplayText><record><rec-number>691</rec-number><foreign-keys><key app="EN" db-id="epzpv0pz5ptrfneaddtxttrw9ea0xdapvvf2" timestamp="1440789740">691</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Altschul, S. F.</author><author>Madden, T. L.</author><author>Schaffer, A. A.</author><author>Zhang, J.</author><author>Zhang, Z.</author><author>Miller, W.</author><author>Lipman, D. J.</author></authors></contributors><auth-address>National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA. altschul@ncbi.nlm.</auth-address><titles><title>Gapped BLAST and PSI-BLAST: a new generation of protein database search programs</title><secondary-title>Nucleic Acids Res</secondary-title></titles><periodical><full-title>Nucleic Acids Res</full-title></periodical><pages>3389-402</pages><volume>25</volume><number>17</number><keywords><keyword>Algorithms</keyword><keyword>Amino Acid Sequence</keyword><keyword>Animal</keyword><keyword>DNA/*chemistry</keyword><keyword>*Databases, Factual</keyword><keyword>Human</keyword><keyword>Molecular Sequence Data</keyword><keyword>Proteins/*chemistry</keyword><keyword>*Sequence Alignment</keyword><keyword>*Software</keyword><keyword>Support, U.S. Gov&apos;t, P.H.S.</keyword></keywords><dates><year>1997</year><pub-dates><date>Sep 1</date></pub-dates></dates><accession-num>9254694</accession-num><urls><related-urls><url>;[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 ADDIN EN.CITE <EndNote><Cite ExcludeYear="1"><Author>Langmead</Author><Year>2012</Year><RecNum>6715</RecNum><DisplayText>[4]</DisplayText><record><rec-number>6715</rec-number><foreign-keys><key app="EN" db-id="fzffee0d8fw0xmeet9655992wpfxp9w2rtzf" timestamp="1442359059">6715</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Langmead, B.</author><author>Salzberg, S. L.</author></authors></contributors><auth-address>Center for Bioinformatics and Computational Biology, Institute for Advanced Computer Studies, University of Maryland, College Park, Maryland, USA. blangmea@jhsph.edu</auth-address><titles><title>Fast gapped-read alignment with Bowtie 2</title><secondary-title>Nat Methods</secondary-title></titles><periodical><full-title>Nat Methods</full-title><abbr-1>Nature methods</abbr-1></periodical><pages>357-9</pages><volume>9</volume><number>4</number><keywords><keyword>*Algorithms</keyword><keyword>Computational Biology/*methods</keyword><keyword>Databases, Genetic</keyword><keyword>Genome, Human/genetics</keyword><keyword>Humans</keyword><keyword>Sequence Alignment/*methods</keyword><keyword>Sequence Analysis, DNA/methods</keyword></keywords><dates><year>2012</year><pub-dates><date>Apr</date></pub-dates></dates><isbn>1548-7105 (Electronic)&#xD;1548-7091 (Linking)</isbn><accession-num>22388286</accession-num><urls><related-urls><url>;[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 PEVuZE5vdGU+PENpdGUgRXhjbHVkZVllYXI9IjEiPjxBdXRob3I+RmVyZW5jenk8L0F1dGhvcj48

WWVhcj4yMDEyPC9ZZWFyPjxSZWNOdW0+MTUyMTwvUmVjTnVtPjxEaXNwbGF5VGV4dD5bNV08L0Rp

c3BsYXlUZXh0PjxyZWNvcmQ+PHJlYy1udW1iZXI+MTUyMTwvcmVjLW51bWJlcj48Zm9yZWlnbi1r

ZXlzPjxrZXkgYXBwPSJFTiIgZGItaWQ9ImVwenB2MHB6NXB0cmZuZWFkZHR4dHRydzllYTB4ZGFw

dnZmMiIgdGltZXN0YW1wPSIxNDQwNzkxMTEzIj4xNTIxPC9rZXk+PC9mb3JlaWduLWtleXM+PHJl

Zi10eXBlIG5hbWU9IkpvdXJuYWwgQXJ0aWNsZSI+MTc8L3JlZi10eXBlPjxjb250cmlidXRvcnM+

PGF1dGhvcnM+PGF1dGhvcj5GZXJlbmN6eSwgTS4gVy48L2F1dGhvcj48YXV0aG9yPk1hcnNoYWxs

LCBMLiBKLjwvYXV0aG9yPjxhdXRob3I+TmVsc29uLCBDLiBELjwvYXV0aG9yPjxhdXRob3I+QXR3

b29kLCBXLiBKLjwvYXV0aG9yPjxhdXRob3I+TmF0aCwgQS48L2F1dGhvcj48YXV0aG9yPktoYWxp

bGksIEsuPC9hdXRob3I+PGF1dGhvcj5NYWpvciwgRS4gTy48L2F1dGhvcj48L2F1dGhvcnM+PC9j

b250cmlidXRvcnM+PGF1dGgtYWRkcmVzcz5MYWJvcmF0b3J5IG9mIE1vbGVjdWxhciBNZWRpY2lu

ZSBhbmQgTmV1cm9zY2llbmNlLCBOYXRpb25hbCBJbnN0aXR1dGUgb2YgTmV1cm9sb2dpY2FsIERp

c29yZGVycyBhbmQgU3Ryb2tlLCBOYXRpb25hbCBJbnN0aXR1dGVzIG9mIEhlYWx0aCwgQmV0aGVz

ZGEsIE1hcnlsYW5kLCBVU0EuPC9hdXRoLWFkZHJlc3M+PHRpdGxlcz48dGl0bGU+TW9sZWN1bGFy

IGJpb2xvZ3ksIGVwaWRlbWlvbG9neSwgYW5kIHBhdGhvZ2VuZXNpcyBvZiBwcm9ncmVzc2l2ZSBt

dWx0aWZvY2FsIGxldWtvZW5jZXBoYWxvcGF0aHksIHRoZSBKQyB2aXJ1cy1pbmR1Y2VkIGRlbXll

bGluYXRpbmcgZGlzZWFzZSBvZiB0aGUgaHVtYW4gYnJhaW48L3RpdGxlPjxzZWNvbmRhcnktdGl0

bGU+Q2xpbiBNaWNyb2Jpb2wgUmV2PC9zZWNvbmRhcnktdGl0bGU+PC90aXRsZXM+PHBlcmlvZGlj

YWw+PGZ1bGwtdGl0bGU+Q2xpbmljYWwgbWljcm9iaW9sb2d5IHJldmlld3M8L2Z1bGwtdGl0bGU+

PGFiYnItMT5DbGluIE1pY3JvYmlvbCBSZXY8L2FiYnItMT48L3BlcmlvZGljYWw+PHBhZ2VzPjQ3

MS01MDY8L3BhZ2VzPjx2b2x1bWU+MjU8L3ZvbHVtZT48bnVtYmVyPjM8L251bWJlcj48a2V5d29y

ZHM+PGtleXdvcmQ+QW50aWJvZGllcywgTW9ub2Nsb25hbCwgSHVtYW5pemVkL2FkdmVyc2UgZWZm

ZWN0czwva2V5d29yZD48a2V5d29yZD5CcmFpbi9wYXRob2xvZ3kvKnZpcm9sb2d5PC9rZXl3b3Jk

PjxrZXl3b3JkPkNvaW5mZWN0aW9uL2VwaWRlbWlvbG9neS9wYXRob2xvZ3kvdmlyb2xvZ3k8L2tl

eXdvcmQ+PGtleXdvcmQ+RGVteWVsaW5hdGluZyBEaXNlYXNlcy9wYXRob2xvZ3kvKnZpcm9sb2d5

PC9rZXl3b3JkPjxrZXl3b3JkPkdlbmUgRXhwcmVzc2lvbiBSZWd1bGF0aW9uLCBWaXJhbDwva2V5

d29yZD48a2V5d29yZD5HZW5vbWUsIFZpcmFsPC9rZXl3b3JkPjxrZXl3b3JkPkhJVi9wYXRob2dl

bmljaXR5PC9rZXl3b3JkPjxrZXl3b3JkPkhJViBJbmZlY3Rpb25zL3BhdGhvbG9neS92aXJvbG9n

eTwva2V5d29yZD48a2V5d29yZD5IdW1hbnM8L2tleXdvcmQ+PGtleXdvcmQ+SW1tdW5lIFJlY29u

c3RpdHV0aW9uIEluZmxhbW1hdG9yeSBTeW5kcm9tZS9wYXRob2xvZ3k8L2tleXdvcmQ+PGtleXdv

cmQ+SkMgVmlydXMvZ2VuZXRpY3MvKnBhdGhvZ2VuaWNpdHkvcGh5c2lvbG9neTwva2V5d29yZD48

a2V5d29yZD5MZXVrb2VuY2VwaGFsb3BhdGh5LCBQcm9ncmVzc2l2ZSBNdWx0aWZvY2FsLyplcGlk

ZW1pb2xvZ3kvcGF0aG9sb2d5L3Zpcm9sb2d5PC9rZXl3b3JkPjxrZXl3b3JkPlJpc2sgRmFjdG9y

czwva2V5d29yZD48a2V5d29yZD5WaXJ1cyBSZXBsaWNhdGlvbi8qZHJ1ZyBlZmZlY3RzPC9rZXl3

b3JkPjwva2V5d29yZHM+PGRhdGVzPjx5ZWFyPjIwMTI8L3llYXI+PHB1Yi1kYXRlcz48ZGF0ZT5K

dWw8L2RhdGU+PC9wdWItZGF0ZXM+PC9kYXRlcz48aXNibj4xMDk4LTY2MTggKEVsZWN0cm9uaWMp

JiN4RDswODkzLTg1MTIgKExpbmtpbmcpPC9pc2JuPjxhY2Nlc3Npb24tbnVtPjIyNzYzNjM1PC9h

Y2Nlc3Npb24tbnVtPjx1cmxzPjxyZWxhdGVkLXVybHM+PHVybD5odHRwOi8vd3d3Lm5jYmkubmxt

Lm5paC5nb3YvcHVibWVkLzIyNzYzNjM1PC91cmw+PC9yZWxhdGVkLXVybHM+PC91cmxzPjxjdXN0

b20yPjM0MTY0OTA8L2N1c3RvbTI+PGVsZWN0cm9uaWMtcmVzb3VyY2UtbnVtPjEwLjExMjgvQ01S

LjA1MDMxLTExPC9lbGVjdHJvbmljLXJlc291cmNlLW51bT48L3JlY29yZD48L0NpdGU+PC9FbmRO

b3RlPgB=

ADDIN EN.CITE PEVuZE5vdGU+PENpdGUgRXhjbHVkZVllYXI9IjEiPjxBdXRob3I+RmVyZW5jenk8L0F1dGhvcj48

WWVhcj4yMDEyPC9ZZWFyPjxSZWNOdW0+MTUyMTwvUmVjTnVtPjxEaXNwbGF5VGV4dD5bNV08L0Rp

c3BsYXlUZXh0PjxyZWNvcmQ+PHJlYy1udW1iZXI+MTUyMTwvcmVjLW51bWJlcj48Zm9yZWlnbi1r

ZXlzPjxrZXkgYXBwPSJFTiIgZGItaWQ9ImVwenB2MHB6NXB0cmZuZWFkZHR4dHRydzllYTB4ZGFw

dnZmMiIgdGltZXN0YW1wPSIxNDQwNzkxMTEzIj4xNTIxPC9rZXk+PC9mb3JlaWduLWtleXM+PHJl

Zi10eXBlIG5hbWU9IkpvdXJuYWwgQXJ0aWNsZSI+MTc8L3JlZi10eXBlPjxjb250cmlidXRvcnM+

PGF1dGhvcnM+PGF1dGhvcj5GZXJlbmN6eSwgTS4gVy48L2F1dGhvcj48YXV0aG9yPk1hcnNoYWxs

LCBMLiBKLjwvYXV0aG9yPjxhdXRob3I+TmVsc29uLCBDLiBELjwvYXV0aG9yPjxhdXRob3I+QXR3

b29kLCBXLiBKLjwvYXV0aG9yPjxhdXRob3I+TmF0aCwgQS48L2F1dGhvcj48YXV0aG9yPktoYWxp

bGksIEsuPC9hdXRob3I+PGF1dGhvcj5NYWpvciwgRS4gTy48L2F1dGhvcj48L2F1dGhvcnM+PC9j

b250cmlidXRvcnM+PGF1dGgtYWRkcmVzcz5MYWJvcmF0b3J5IG9mIE1vbGVjdWxhciBNZWRpY2lu

ZSBhbmQgTmV1cm9zY2llbmNlLCBOYXRpb25hbCBJbnN0aXR1dGUgb2YgTmV1cm9sb2dpY2FsIERp

c29yZGVycyBhbmQgU3Ryb2tlLCBOYXRpb25hbCBJbnN0aXR1dGVzIG9mIEhlYWx0aCwgQmV0aGVz

ZGEsIE1hcnlsYW5kLCBVU0EuPC9hdXRoLWFkZHJlc3M+PHRpdGxlcz48dGl0bGU+TW9sZWN1bGFy

IGJpb2xvZ3ksIGVwaWRlbWlvbG9neSwgYW5kIHBhdGhvZ2VuZXNpcyBvZiBwcm9ncmVzc2l2ZSBt

dWx0aWZvY2FsIGxldWtvZW5jZXBoYWxvcGF0aHksIHRoZSBKQyB2aXJ1cy1pbmR1Y2VkIGRlbXll

bGluYXRpbmcgZGlzZWFzZSBvZiB0aGUgaHVtYW4gYnJhaW48L3RpdGxlPjxzZWNvbmRhcnktdGl0

bGU+Q2xpbiBNaWNyb2Jpb2wgUmV2PC9zZWNvbmRhcnktdGl0bGU+PC90aXRsZXM+PHBlcmlvZGlj

YWw+PGZ1bGwtdGl0bGU+Q2xpbmljYWwgbWljcm9iaW9sb2d5IHJldmlld3M8L2Z1bGwtdGl0bGU+

PGFiYnItMT5DbGluIE1pY3JvYmlvbCBSZXY8L2FiYnItMT48L3BlcmlvZGljYWw+PHBhZ2VzPjQ3

MS01MDY8L3BhZ2VzPjx2b2x1bWU+MjU8L3ZvbHVtZT48bnVtYmVyPjM8L251bWJlcj48a2V5d29y

ZHM+PGtleXdvcmQ+QW50aWJvZGllcywgTW9ub2Nsb25hbCwgSHVtYW5pemVkL2FkdmVyc2UgZWZm

ZWN0czwva2V5d29yZD48a2V5d29yZD5CcmFpbi9wYXRob2xvZ3kvKnZpcm9sb2d5PC9rZXl3b3Jk

PjxrZXl3b3JkPkNvaW5mZWN0aW9uL2VwaWRlbWlvbG9neS9wYXRob2xvZ3kvdmlyb2xvZ3k8L2tl

eXdvcmQ+PGtleXdvcmQ+RGVteWVsaW5hdGluZyBEaXNlYXNlcy9wYXRob2xvZ3kvKnZpcm9sb2d5

PC9rZXl3b3JkPjxrZXl3b3JkPkdlbmUgRXhwcmVzc2lvbiBSZWd1bGF0aW9uLCBWaXJhbDwva2V5

d29yZD48a2V5d29yZD5HZW5vbWUsIFZpcmFsPC9rZXl3b3JkPjxrZXl3b3JkPkhJVi9wYXRob2dl

bmljaXR5PC9rZXl3b3JkPjxrZXl3b3JkPkhJViBJbmZlY3Rpb25zL3BhdGhvbG9neS92aXJvbG9n

eTwva2V5d29yZD48a2V5d29yZD5IdW1hbnM8L2tleXdvcmQ+PGtleXdvcmQ+SW1tdW5lIFJlY29u

c3RpdHV0aW9uIEluZmxhbW1hdG9yeSBTeW5kcm9tZS9wYXRob2xvZ3k8L2tleXdvcmQ+PGtleXdv

cmQ+SkMgVmlydXMvZ2VuZXRpY3MvKnBhdGhvZ2VuaWNpdHkvcGh5c2lvbG9neTwva2V5d29yZD48

a2V5d29yZD5MZXVrb2VuY2VwaGFsb3BhdGh5LCBQcm9ncmVzc2l2ZSBNdWx0aWZvY2FsLyplcGlk

ZW1pb2xvZ3kvcGF0aG9sb2d5L3Zpcm9sb2d5PC9rZXl3b3JkPjxrZXl3b3JkPlJpc2sgRmFjdG9y

czwva2V5d29yZD48a2V5d29yZD5WaXJ1cyBSZXBsaWNhdGlvbi8qZHJ1ZyBlZmZlY3RzPC9rZXl3

b3JkPjwva2V5d29yZHM+PGRhdGVzPjx5ZWFyPjIwMTI8L3llYXI+PHB1Yi1kYXRlcz48ZGF0ZT5K

dWw8L2RhdGU+PC9wdWItZGF0ZXM+PC9kYXRlcz48aXNibj4xMDk4LTY2MTggKEVsZWN0cm9uaWMp

JiN4RDswODkzLTg1MTIgKExpbmtpbmcpPC9pc2JuPjxhY2Nlc3Npb24tbnVtPjIyNzYzNjM1PC9h

Y2Nlc3Npb24tbnVtPjx1cmxzPjxyZWxhdGVkLXVybHM+PHVybD5odHRwOi8vd3d3Lm5jYmkubmxt

Lm5paC5nb3YvcHVibWVkLzIyNzYzNjM1PC91cmw+PC9yZWxhdGVkLXVybHM+PC91cmxzPjxjdXN0

b20yPjM0MTY0OTA8L2N1c3RvbTI+PGVsZWN0cm9uaWMtcmVzb3VyY2UtbnVtPjEwLjExMjgvQ01S

LjA1MDMxLTExPC9lbGVjdHJvbmljLXJlc291cmNlLW51bT48L3JlY29yZD48L0NpdGU+PC9FbmRO

b3RlPgB=

ADDIN EN.CITE.DATA [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 PEVuZE5vdGU+PENpdGUgRXhjbHVkZVllYXI9IjEiPjxBdXRob3I+S2hhbGlsPC9BdXRob3I+PFll

YXI+MjAwODwvWWVhcj48UmVjTnVtPjE2MjI8L1JlY051bT48RGlzcGxheVRleHQ+WzZdPC9EaXNw

bGF5VGV4dD48cmVjb3JkPjxyZWMtbnVtYmVyPjE2MjI8L3JlYy1udW1iZXI+PGZvcmVpZ24ta2V5

cz48a2V5IGFwcD0iRU4iIGRiLWlkPSJlcHpwdjBwejVwdHJmbmVhZGR0eHR0cnc5ZWEweGRhcHZ2

ZjIiIHRpbWVzdGFtcD0iMTQ0MDc5MTgxNiI+MTYyMjwva2V5PjwvZm9yZWlnbi1rZXlzPjxyZWYt

dHlwZSBuYW1lPSJKb3VybmFsIEFydGljbGUiPjE3PC9yZWYtdHlwZT48Y29udHJpYnV0b3JzPjxh

dXRob3JzPjxhdXRob3I+S2hhbGlsLCBNLjwvYXV0aG9yPjxhdXRob3I+RW56aW5nZXIsIEMuPC9h

dXRob3I+PGF1dGhvcj5XYWxsbmVyLUJsYXplaywgTS48L2F1dGhvcj48YXV0aG9yPlNjYXJwYXRl

dHRpLCBNLjwvYXV0aG9yPjxhdXRob3I+QmFydGgsIEEuPC9hdXRob3I+PGF1dGhvcj5Ib3JuLCBT

LjwvYXV0aG9yPjxhdXRob3I+UmVpdGVyLCBHLjwvYXV0aG9yPjwvYXV0aG9ycz48L2NvbnRyaWJ1

dG9ycz48YXV0aC1hZGRyZXNzPkRlcGFydG1lbnQgb2YgTmV1cm9sb2d5LCBNZWRpY2FsIFVuaXZl

cnNpdHkgR3JheiwgR3JheiwgQXVzdHJpYS4gTWVkaWNhbCBVbml2ZXJzaXR5IEdyYXosIEdyYXos

IEF1c3RyaWE8L2F1dGgtYWRkcmVzcz48dGl0bGVzPjx0aXRsZT5FcHN0ZWluLUJhcnIgdmlydXMg

ZW5jZXBoYWxpdGlzIHByZXNlbnRpbmcgd2l0aCBhIHR1bW9yLWxpa2UgbGVzaW9uIGluIGFuIGlt

bXVub3N1cHByZXNzZWQgdHJhbnNwbGFudCByZWNpcGllbnQ8L3RpdGxlPjxzZWNvbmRhcnktdGl0

bGU+SiBOZXVyb3Zpcm9sPC9zZWNvbmRhcnktdGl0bGU+PC90aXRsZXM+PHBlcmlvZGljYWw+PGZ1

bGwtdGl0bGU+SiBOZXVyb3Zpcm9sPC9mdWxsLXRpdGxlPjwvcGVyaW9kaWNhbD48cGFnZXM+NTc0

LTg8L3BhZ2VzPjx2b2x1bWU+MTQ8L3ZvbHVtZT48bnVtYmVyPjY8L251bWJlcj48a2V5d29yZHM+

PGtleXdvcmQ+QW50aXZpcmFsIEFnZW50cy90aGVyYXBldXRpYyB1c2U8L2tleXdvcmQ+PGtleXdv

cmQ+QnJhaW4gTmVvcGxhc21zL2RpYWdub3Npcy9zdXJnZXJ5PC9rZXl3b3JkPjxrZXl3b3JkPkRO

QSwgVmlyYWwvYmxvb2QvY2VyZWJyb3NwaW5hbCBmbHVpZDwva2V5d29yZD48a2V5d29yZD5EaWFn

bm9zaXMsIERpZmZlcmVudGlhbDwva2V5d29yZD48a2V5d29yZD5FbmNlcGhhbGl0aXMsIFZpcmFs

LypkaWFnbm9zaXMvZHJ1ZyB0aGVyYXB5PC9rZXl3b3JkPjxrZXl3b3JkPkVwc3RlaW4tQmFyciBW

aXJ1cyBJbmZlY3Rpb25zLypkaWFnbm9zaXMvZHJ1ZyB0aGVyYXB5L3BhdGhvbG9neTwva2V5d29y

ZD48a2V5d29yZD5GZW1hbGU8L2tleXdvcmQ+PGtleXdvcmQ+R2FuY2ljbG92aXIvdGhlcmFwZXV0

aWMgdXNlPC9rZXl3b3JkPjxrZXl3b3JkPkdsaW9tYS9kaWFnbm9zaXMvc3VyZ2VyeTwva2V5d29y

ZD48a2V5d29yZD5IZXJwZXN2aXJ1cyA0LCBIdW1hbi9nZW5ldGljcy8qaXNvbGF0aW9uICZhbXA7

IHB1cmlmaWNhdGlvbjwva2V5d29yZD48a2V5d29yZD5IdW1hbnM8L2tleXdvcmQ+PGtleXdvcmQ+

SW1tdW5vY29tcHJvbWlzZWQgSG9zdDwva2V5d29yZD48a2V5d29yZD5JbW11bm9zdXBwcmVzc2l2

ZSBBZ2VudHMvYWR2ZXJzZSBlZmZlY3RzL3RoZXJhcGV1dGljIHVzZTwva2V5d29yZD48a2V5d29y

ZD5LaWRuZXkgVHJhbnNwbGFudGF0aW9uL2FkdmVyc2UgZWZmZWN0cy9pbW11bm9sb2d5PC9rZXl3

b3JkPjxrZXl3b3JkPk1hZ25ldGljIFJlc29uYW5jZSBJbWFnaW5nPC9rZXl3b3JkPjxrZXl3b3Jk

Pk1pZGRsZSBBZ2VkPC9rZXl3b3JkPjxrZXl3b3JkPlBhbmNyZWFzIFRyYW5zcGxhbnRhdGlvbi9h

ZHZlcnNlIGVmZmVjdHMvaW1tdW5vbG9neTwva2V5d29yZD48a2V5d29yZD5WaXN1YWwgUGF0aHdh

eXMvcGF0aG9sb2d5PC9rZXl3b3JkPjxrZXl3b3JkPldpdGhob2xkaW5nIFRyZWF0bWVudDwva2V5

d29yZD48L2tleXdvcmRzPjxkYXRlcz48eWVhcj4yMDA4PC95ZWFyPjxwdWItZGF0ZXM+PGRhdGU+

Tm92PC9kYXRlPjwvcHViLWRhdGVzPjwvZGF0ZXM+PGlzYm4+MTUzOC0yNDQzIChFbGVjdHJvbmlj

KSYjeEQ7MTM1NS0wMjg0IChMaW5raW5nKTwvaXNibj48YWNjZXNzaW9uLW51bT4xODk5MTA3MDwv

YWNjZXNzaW9uLW51bT48dXJscz48cmVsYXRlZC11cmxzPjx1cmw+aHR0cDovL3d3dy5uY2JpLm5s

bS5uaWguZ292L3B1Ym1lZC8xODk5MTA3MDwvdXJsPjwvcmVsYXRlZC11cmxzPjwvdXJscz48ZWxl

Y3Ryb25pYy1yZXNvdXJjZS1udW0+MTAuMTA4MC8xMzU1MDI4MDgwMjM0NTcxNTwvZWxlY3Ryb25p

Yy1yZXNvdXJjZS1udW0+PC9yZWNvcmQ+PC9DaXRlPjwvRW5kTm90ZT5=

ADDIN EN.CITE PEVuZE5vdGU+PENpdGUgRXhjbHVkZVllYXI9IjEiPjxBdXRob3I+S2hhbGlsPC9BdXRob3I+PFll

YXI+MjAwODwvWWVhcj48UmVjTnVtPjE2MjI8L1JlY051bT48RGlzcGxheVRleHQ+WzZdPC9EaXNw

bGF5VGV4dD48cmVjb3JkPjxyZWMtbnVtYmVyPjE2MjI8L3JlYy1udW1iZXI+PGZvcmVpZ24ta2V5

cz48a2V5IGFwcD0iRU4iIGRiLWlkPSJlcHpwdjBwejVwdHJmbmVhZGR0eHR0cnc5ZWEweGRhcHZ2

ZjIiIHRpbWVzdGFtcD0iMTQ0MDc5MTgxNiI+MTYyMjwva2V5PjwvZm9yZWlnbi1rZXlzPjxyZWYt

dHlwZSBuYW1lPSJKb3VybmFsIEFydGljbGUiPjE3PC9yZWYtdHlwZT48Y29udHJpYnV0b3JzPjxh

dXRob3JzPjxhdXRob3I+S2hhbGlsLCBNLjwvYXV0aG9yPjxhdXRob3I+RW56aW5nZXIsIEMuPC9h

dXRob3I+PGF1dGhvcj5XYWxsbmVyLUJsYXplaywgTS48L2F1dGhvcj48YXV0aG9yPlNjYXJwYXRl

dHRpLCBNLjwvYXV0aG9yPjxhdXRob3I+QmFydGgsIEEuPC9hdXRob3I+PGF1dGhvcj5Ib3JuLCBT

LjwvYXV0aG9yPjxhdXRob3I+UmVpdGVyLCBHLjwvYXV0aG9yPjwvYXV0aG9ycz48L2NvbnRyaWJ1

dG9ycz48YXV0aC1hZGRyZXNzPkRlcGFydG1lbnQgb2YgTmV1cm9sb2d5LCBNZWRpY2FsIFVuaXZl

cnNpdHkgR3JheiwgR3JheiwgQXVzdHJpYS4gTWVkaWNhbCBVbml2ZXJzaXR5IEdyYXosIEdyYXos

IEF1c3RyaWE8L2F1dGgtYWRkcmVzcz48dGl0bGVzPjx0aXRsZT5FcHN0ZWluLUJhcnIgdmlydXMg

ZW5jZXBoYWxpdGlzIHByZXNlbnRpbmcgd2l0aCBhIHR1bW9yLWxpa2UgbGVzaW9uIGluIGFuIGlt

bXVub3N1cHByZXNzZWQgdHJhbnNwbGFudCByZWNpcGllbnQ8L3RpdGxlPjxzZWNvbmRhcnktdGl0

bGU+SiBOZXVyb3Zpcm9sPC9zZWNvbmRhcnktdGl0bGU+PC90aXRsZXM+PHBlcmlvZGljYWw+PGZ1

bGwtdGl0bGU+SiBOZXVyb3Zpcm9sPC9mdWxsLXRpdGxlPjwvcGVyaW9kaWNhbD48cGFnZXM+NTc0

LTg8L3BhZ2VzPjx2b2x1bWU+MTQ8L3ZvbHVtZT48bnVtYmVyPjY8L251bWJlcj48a2V5d29yZHM+

PGtleXdvcmQ+QW50aXZpcmFsIEFnZW50cy90aGVyYXBldXRpYyB1c2U8L2tleXdvcmQ+PGtleXdv

cmQ+QnJhaW4gTmVvcGxhc21zL2RpYWdub3Npcy9zdXJnZXJ5PC9rZXl3b3JkPjxrZXl3b3JkPkRO

QSwgVmlyYWwvYmxvb2QvY2VyZWJyb3NwaW5hbCBmbHVpZDwva2V5d29yZD48a2V5d29yZD5EaWFn

bm9zaXMsIERpZmZlcmVudGlhbDwva2V5d29yZD48a2V5d29yZD5FbmNlcGhhbGl0aXMsIFZpcmFs

LypkaWFnbm9zaXMvZHJ1ZyB0aGVyYXB5PC9rZXl3b3JkPjxrZXl3b3JkPkVwc3RlaW4tQmFyciBW

aXJ1cyBJbmZlY3Rpb25zLypkaWFnbm9zaXMvZHJ1ZyB0aGVyYXB5L3BhdGhvbG9neTwva2V5d29y

ZD48a2V5d29yZD5GZW1hbGU8L2tleXdvcmQ+PGtleXdvcmQ+R2FuY2ljbG92aXIvdGhlcmFwZXV0

aWMgdXNlPC9rZXl3b3JkPjxrZXl3b3JkPkdsaW9tYS9kaWFnbm9zaXMvc3VyZ2VyeTwva2V5d29y

ZD48a2V5d29yZD5IZXJwZXN2aXJ1cyA0LCBIdW1hbi9nZW5ldGljcy8qaXNvbGF0aW9uICZhbXA7

IHB1cmlmaWNhdGlvbjwva2V5d29yZD48a2V5d29yZD5IdW1hbnM8L2tleXdvcmQ+PGtleXdvcmQ+

SW1tdW5vY29tcHJvbWlzZWQgSG9zdDwva2V5d29yZD48a2V5d29yZD5JbW11bm9zdXBwcmVzc2l2

ZSBBZ2VudHMvYWR2ZXJzZSBlZmZlY3RzL3RoZXJhcGV1dGljIHVzZTwva2V5d29yZD48a2V5d29y

ZD5LaWRuZXkgVHJhbnNwbGFudGF0aW9uL2FkdmVyc2UgZWZmZWN0cy9pbW11bm9sb2d5PC9rZXl3

b3JkPjxrZXl3b3JkPk1hZ25ldGljIFJlc29uYW5jZSBJbWFnaW5nPC9rZXl3b3JkPjxrZXl3b3Jk

Pk1pZGRsZSBBZ2VkPC9rZXl3b3JkPjxrZXl3b3JkPlBhbmNyZWFzIFRyYW5zcGxhbnRhdGlvbi9h

ZHZlcnNlIGVmZmVjdHMvaW1tdW5vbG9neTwva2V5d29yZD48a2V5d29yZD5WaXN1YWwgUGF0aHdh

eXMvcGF0aG9sb2d5PC9rZXl3b3JkPjxrZXl3b3JkPldpdGhob2xkaW5nIFRyZWF0bWVudDwva2V5

d29yZD48L2tleXdvcmRzPjxkYXRlcz48eWVhcj4yMDA4PC95ZWFyPjxwdWItZGF0ZXM+PGRhdGU+

Tm92PC9kYXRlPjwvcHViLWRhdGVzPjwvZGF0ZXM+PGlzYm4+MTUzOC0yNDQzIChFbGVjdHJvbmlj

KSYjeEQ7MTM1NS0wMjg0IChMaW5raW5nKTwvaXNibj48YWNjZXNzaW9uLW51bT4xODk5MTA3MDwv

YWNjZXNzaW9uLW51bT48dXJscz48cmVsYXRlZC11cmxzPjx1cmw+aHR0cDovL3d3dy5uY2JpLm5s

bS5uaWguZ292L3B1Ym1lZC8xODk5MTA3MDwvdXJsPjwvcmVsYXRlZC11cmxzPjwvdXJscz48ZWxl

Y3Ryb25pYy1yZXNvdXJjZS1udW0+MTAuMTA4MC8xMzU1MDI4MDgwMjM0NTcxNTwvZWxlY3Ryb25p

Yy1yZXNvdXJjZS1udW0+PC9yZWNvcmQ+PC9DaXRlPjwvRW5kTm90ZT5=

ADDIN EN.CITE.DATA [6] of EBV-induced brain lesions in an immunosuppressed patient following organ transplantation.Cases with indeterminate pathogen identification but possible infectionPatient 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 PEVuZE5vdGU+PENpdGUgRXhjbHVkZVllYXI9IjEiPjxBdXRob3I+UGFyZG88L0F1dGhvcj48WWVh

cj4yMDE0PC9ZZWFyPjxSZWNOdW0+Mzk0NDwvUmVjTnVtPjxEaXNwbGF5VGV4dD5bN108L0Rpc3Bs

YXlUZXh0PjxyZWNvcmQ+PHJlYy1udW1iZXI+Mzk0NDwvcmVjLW51bWJlcj48Zm9yZWlnbi1rZXlz

PjxrZXkgYXBwPSJFTiIgZGItaWQ9ImZ6ZmZlZTBkOGZ3MHhtZWV0OTY1NTk5MndwZnhwOXcycnR6

ZiIgdGltZXN0YW1wPSIxNDIwOTQyNzMyIj4zOTQ0PC9rZXk+PC9mb3JlaWduLWtleXM+PHJlZi10

eXBlIG5hbWU9IkpvdXJuYWwgQXJ0aWNsZSI+MTc8L3JlZi10eXBlPjxjb250cmlidXRvcnM+PGF1

dGhvcnM+PGF1dGhvcj5QYXJkbywgQy4gQS48L2F1dGhvcj48YXV0aG9yPk5hYmJvdXQsIFIuPC9h

dXRob3I+PGF1dGhvcj5HYWxhbm9wb3Vsb3UsIEEuIFMuPC9hdXRob3I+PC9hdXRob3JzPjwvY29u

dHJpYnV0b3JzPjxhdXRoLWFkZHJlc3M+RGVwYXJ0bWVudCBvZiBOZXVyb2xvZ3ksIERpdmlzaW9u

IG9mIE5ldXJvaW1tdW5vbG9neSBhbmQgTmV1cm9pbmZlY3Rpb3VzIERpc29yZGVycywgQ2VudGVy

IGZvciBQZWRpYXRyaWMgUmFzbXVzc2VuIFN5bmRyb21lLCBKb2hucyBIb3BraW5zIFVuaXZlcnNp

dHkgU2Nob29sIG9mIE1lZGljaW5lLCBCYWx0aW1vcmUsIE1ELCBVU0EsIGNwYXJkb3YxQGpobWku

ZWR1LjwvYXV0aC1hZGRyZXNzPjx0aXRsZXM+PHRpdGxlPk1lY2hhbmlzbXMgb2YgZXBpbGVwdG9n

ZW5lc2lzIGluIHBlZGlhdHJpYyBlcGlsZXB0aWMgc3luZHJvbWVzOiBSYXNtdXNzZW4gZW5jZXBo

YWxpdGlzLCBpbmZhbnRpbGUgc3Bhc21zLCBhbmQgZmVicmlsZSBpbmZlY3Rpb24tcmVsYXRlZCBl

cGlsZXBzeSBzeW5kcm9tZSAoRklSRVMpPC90aXRsZT48c2Vjb25kYXJ5LXRpdGxlPk5ldXJvdGhl

cmFwZXV0aWNzPC9zZWNvbmRhcnktdGl0bGU+PGFsdC10aXRsZT5OZXVyb3RoZXJhcGV1dGljcyA6

IHRoZSBqb3VybmFsIG9mIHRoZSBBbWVyaWNhbiBTb2NpZXR5IGZvciBFeHBlcmltZW50YWwgTmV1

cm9UaGVyYXBldXRpY3M8L2FsdC10aXRsZT48L3RpdGxlcz48cGVyaW9kaWNhbD48ZnVsbC10aXRs

ZT5OZXVyb3RoZXJhcGV1dGljczwvZnVsbC10aXRsZT48YWJici0xPk5ldXJvdGhlcmFwZXV0aWNz

IDogdGhlIGpvdXJuYWwgb2YgdGhlIEFtZXJpY2FuIFNvY2lldHkgZm9yIEV4cGVyaW1lbnRhbCBO

ZXVyb1RoZXJhcGV1dGljczwvYWJici0xPjwvcGVyaW9kaWNhbD48YWx0LXBlcmlvZGljYWw+PGZ1

bGwtdGl0bGU+TmV1cm90aGVyYXBldXRpY3M8L2Z1bGwtdGl0bGU+PGFiYnItMT5OZXVyb3RoZXJh

cGV1dGljcyA6IHRoZSBqb3VybmFsIG9mIHRoZSBBbWVyaWNhbiBTb2NpZXR5IGZvciBFeHBlcmlt

ZW50YWwgTmV1cm9UaGVyYXBldXRpY3M8L2FiYnItMT48L2FsdC1wZXJpb2RpY2FsPjxwYWdlcz4y

OTctMzEwPC9wYWdlcz48dm9sdW1lPjExPC92b2x1bWU+PG51bWJlcj4yPC9udW1iZXI+PGVkaXRp

b24+MjAxNC8wMy8xOTwvZWRpdGlvbj48a2V5d29yZHM+PGtleXdvcmQ+RW5jZXBoYWxpdGlzLypj

b21wbGljYXRpb25zL2ltbXVub2xvZ3k8L2tleXdvcmQ+PGtleXdvcmQ+RXBpbGVwc3kvKmV0aW9s

b2d5L2ltbXVub2xvZ3k8L2tleXdvcmQ+PGtleXdvcmQ+SHVtYW5zPC9rZXl3b3JkPjxrZXl3b3Jk

PkluZmFudDwva2V5d29yZD48a2V5d29yZD5JbmZlY3Rpb24vY29tcGxpY2F0aW9uczwva2V5d29y

ZD48a2V5d29yZD5TZWl6dXJlcywgRmVicmlsZS8qY29tcGxpY2F0aW9ucy9pbW11bm9sb2d5PC9r

ZXl3b3JkPjxrZXl3b3JkPlNwYXNtcywgSW5mYW50aWxlLypjb21wbGljYXRpb25zL2ltbXVub2xv

Z3k8L2tleXdvcmQ+PGtleXdvcmQ+U3luZHJvbWU8L2tleXdvcmQ+PC9rZXl3b3Jkcz48ZGF0ZXM+

PHllYXI+MjAxNDwveWVhcj48cHViLWRhdGVzPjxkYXRlPkFwcjwvZGF0ZT48L3B1Yi1kYXRlcz48

L2RhdGVzPjxpc2JuPjE4NzgtNzQ3OTwvaXNibj48YWNjZXNzaW9uLW51bT4yNDYzOTM3NTwvYWNj

ZXNzaW9uLW51bT48dXJscz48L3VybHM+PGN1c3RvbTI+UG1jMzk5NjExNjwvY3VzdG9tMj48ZWxl

Y3Ryb25pYy1yZXNvdXJjZS1udW0+MTAuMTAwNy9zMTMzMTEtMDE0LTAyNjUtMjwvZWxlY3Ryb25p

Yy1yZXNvdXJjZS1udW0+PHJlbW90ZS1kYXRhYmFzZS1wcm92aWRlcj5OTE08L3JlbW90ZS1kYXRh

YmFzZS1wcm92aWRlcj48bGFuZ3VhZ2U+ZW5nPC9sYW5ndWFnZT48L3JlY29yZD48L0NpdGU+PC9F

bmROb3RlPn==

ADDIN EN.CITE PEVuZE5vdGU+PENpdGUgRXhjbHVkZVllYXI9IjEiPjxBdXRob3I+UGFyZG88L0F1dGhvcj48WWVh

cj4yMDE0PC9ZZWFyPjxSZWNOdW0+Mzk0NDwvUmVjTnVtPjxEaXNwbGF5VGV4dD5bN108L0Rpc3Bs

YXlUZXh0PjxyZWNvcmQ+PHJlYy1udW1iZXI+Mzk0NDwvcmVjLW51bWJlcj48Zm9yZWlnbi1rZXlz

PjxrZXkgYXBwPSJFTiIgZGItaWQ9ImZ6ZmZlZTBkOGZ3MHhtZWV0OTY1NTk5MndwZnhwOXcycnR6

ZiIgdGltZXN0YW1wPSIxNDIwOTQyNzMyIj4zOTQ0PC9rZXk+PC9mb3JlaWduLWtleXM+PHJlZi10

eXBlIG5hbWU9IkpvdXJuYWwgQXJ0aWNsZSI+MTc8L3JlZi10eXBlPjxjb250cmlidXRvcnM+PGF1

dGhvcnM+PGF1dGhvcj5QYXJkbywgQy4gQS48L2F1dGhvcj48YXV0aG9yPk5hYmJvdXQsIFIuPC9h

dXRob3I+PGF1dGhvcj5HYWxhbm9wb3Vsb3UsIEEuIFMuPC9hdXRob3I+PC9hdXRob3JzPjwvY29u

dHJpYnV0b3JzPjxhdXRoLWFkZHJlc3M+RGVwYXJ0bWVudCBvZiBOZXVyb2xvZ3ksIERpdmlzaW9u

IG9mIE5ldXJvaW1tdW5vbG9neSBhbmQgTmV1cm9pbmZlY3Rpb3VzIERpc29yZGVycywgQ2VudGVy

IGZvciBQZWRpYXRyaWMgUmFzbXVzc2VuIFN5bmRyb21lLCBKb2hucyBIb3BraW5zIFVuaXZlcnNp

dHkgU2Nob29sIG9mIE1lZGljaW5lLCBCYWx0aW1vcmUsIE1ELCBVU0EsIGNwYXJkb3YxQGpobWku

ZWR1LjwvYXV0aC1hZGRyZXNzPjx0aXRsZXM+PHRpdGxlPk1lY2hhbmlzbXMgb2YgZXBpbGVwdG9n

ZW5lc2lzIGluIHBlZGlhdHJpYyBlcGlsZXB0aWMgc3luZHJvbWVzOiBSYXNtdXNzZW4gZW5jZXBo

YWxpdGlzLCBpbmZhbnRpbGUgc3Bhc21zLCBhbmQgZmVicmlsZSBpbmZlY3Rpb24tcmVsYXRlZCBl

cGlsZXBzeSBzeW5kcm9tZSAoRklSRVMpPC90aXRsZT48c2Vjb25kYXJ5LXRpdGxlPk5ldXJvdGhl

cmFwZXV0aWNzPC9zZWNvbmRhcnktdGl0bGU+PGFsdC10aXRsZT5OZXVyb3RoZXJhcGV1dGljcyA6

IHRoZSBqb3VybmFsIG9mIHRoZSBBbWVyaWNhbiBTb2NpZXR5IGZvciBFeHBlcmltZW50YWwgTmV1

cm9UaGVyYXBldXRpY3M8L2FsdC10aXRsZT48L3RpdGxlcz48cGVyaW9kaWNhbD48ZnVsbC10aXRs

ZT5OZXVyb3RoZXJhcGV1dGljczwvZnVsbC10aXRsZT48YWJici0xPk5ldXJvdGhlcmFwZXV0aWNz

IDogdGhlIGpvdXJuYWwgb2YgdGhlIEFtZXJpY2FuIFNvY2lldHkgZm9yIEV4cGVyaW1lbnRhbCBO

ZXVyb1RoZXJhcGV1dGljczwvYWJici0xPjwvcGVyaW9kaWNhbD48YWx0LXBlcmlvZGljYWw+PGZ1

bGwtdGl0bGU+TmV1cm90aGVyYXBldXRpY3M8L2Z1bGwtdGl0bGU+PGFiYnItMT5OZXVyb3RoZXJh

cGV1dGljcyA6IHRoZSBqb3VybmFsIG9mIHRoZSBBbWVyaWNhbiBTb2NpZXR5IGZvciBFeHBlcmlt

ZW50YWwgTmV1cm9UaGVyYXBldXRpY3M8L2FiYnItMT48L2FsdC1wZXJpb2RpY2FsPjxwYWdlcz4y

OTctMzEwPC9wYWdlcz48dm9sdW1lPjExPC92b2x1bWU+PG51bWJlcj4yPC9udW1iZXI+PGVkaXRp

b24+MjAxNC8wMy8xOTwvZWRpdGlvbj48a2V5d29yZHM+PGtleXdvcmQ+RW5jZXBoYWxpdGlzLypj

b21wbGljYXRpb25zL2ltbXVub2xvZ3k8L2tleXdvcmQ+PGtleXdvcmQ+RXBpbGVwc3kvKmV0aW9s

b2d5L2ltbXVub2xvZ3k8L2tleXdvcmQ+PGtleXdvcmQ+SHVtYW5zPC9rZXl3b3JkPjxrZXl3b3Jk

PkluZmFudDwva2V5d29yZD48a2V5d29yZD5JbmZlY3Rpb24vY29tcGxpY2F0aW9uczwva2V5d29y

ZD48a2V5d29yZD5TZWl6dXJlcywgRmVicmlsZS8qY29tcGxpY2F0aW9ucy9pbW11bm9sb2d5PC9r

ZXl3b3JkPjxrZXl3b3JkPlNwYXNtcywgSW5mYW50aWxlLypjb21wbGljYXRpb25zL2ltbXVub2xv

Z3k8L2tleXdvcmQ+PGtleXdvcmQ+U3luZHJvbWU8L2tleXdvcmQ+PC9rZXl3b3Jkcz48ZGF0ZXM+

PHllYXI+MjAxNDwveWVhcj48cHViLWRhdGVzPjxkYXRlPkFwcjwvZGF0ZT48L3B1Yi1kYXRlcz48

L2RhdGVzPjxpc2JuPjE4NzgtNzQ3OTwvaXNibj48YWNjZXNzaW9uLW51bT4yNDYzOTM3NTwvYWNj

ZXNzaW9uLW51bT48dXJscz48L3VybHM+PGN1c3RvbTI+UG1jMzk5NjExNjwvY3VzdG9tMj48ZWxl

Y3Ryb25pYy1yZXNvdXJjZS1udW0+MTAuMTAwNy9zMTMzMTEtMDE0LTAyNjUtMjwvZWxlY3Ryb25p

Yy1yZXNvdXJjZS1udW0+PHJlbW90ZS1kYXRhYmFzZS1wcm92aWRlcj5OTE08L3JlbW90ZS1kYXRh

YmFzZS1wcm92aWRlcj48bGFuZ3VhZ2U+ZW5nPC9sYW5ndWFnZT48L3JlY29yZD48L0NpdGU+PC9F

bmROb3RlPn==

ADDIN EN.CITE.DATA [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 ADDIN EN.CITE <EndNote><Cite ExcludeYear="1"><Author>Maginnis</Author><Year>2009</Year><RecNum>6713</RecNum><DisplayText>[8]</DisplayText><record><rec-number>6713</rec-number><foreign-keys><key app="EN" db-id="fzffee0d8fw0xmeet9655992wpfxp9w2rtzf" timestamp="1442358934">6713</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Maginnis, M. S.</author><author>Atwood, W. J.</author></authors></contributors><auth-address>Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02903, USA.</auth-address><titles><title>JC virus: an oncogenic virus in animals and humans?</title><secondary-title>Semin Cancer Biol</secondary-title></titles><periodical><full-title>Semin Cancer Biol</full-title></periodical><pages>261-9</pages><volume>19</volume><number>4</number><keywords><keyword>Animals</keyword><keyword>Humans</keyword><keyword>JC Virus/*physiology</keyword><keyword>Leukoencephalopathy, Progressive Multifocal/virology</keyword><keyword>Neoplasms/*virology</keyword><keyword>Polyomavirus Infections/*complications</keyword><keyword>Tumor Virus Infections/*complications</keyword></keywords><dates><year>2009</year><pub-dates><date>Aug</date></pub-dates></dates><isbn>1096-3650 (Electronic)&#xD;1044-579X (Linking)</isbn><accession-num>19505654</accession-num><urls><related-urls><url>;[8], and it may infect astrocytes in humans ADDIN EN.CITE <EndNote><Cite ExcludeYear="1"><Author>Sofroniew</Author><Year>2010</Year><RecNum>6711</RecNum><DisplayText>[9]</DisplayText><record><rec-number>6711</rec-number><foreign-keys><key app="EN" db-id="fzffee0d8fw0xmeet9655992wpfxp9w2rtzf" timestamp="1442358825">6711</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Sofroniew, M. V.</author><author>Vinters, H. V.</author></authors></contributors><auth-address>Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095-1763, USA. sofroniew@mednet.ucla.edu</auth-address><titles><title>Astrocytes: biology and pathology</title><secondary-title>Acta Neuropathol</secondary-title></titles><periodical><full-title>Acta Neuropathol</full-title><abbr-1>Acta neuropathologica</abbr-1></periodical><pages>7-35</pages><volume>119</volume><number>1</number><keywords><keyword>Animals</keyword><keyword>Astrocytes/*pathology/*physiology</keyword><keyword>Central Nervous System/pathology/physiology/physiopathology</keyword><keyword>Central Nervous System Diseases/pathology/physiopathology</keyword><keyword>Humans</keyword><keyword>Models, Neurological</keyword></keywords><dates><year>2010</year><pub-dates><date>Jan</date></pub-dates></dates><isbn>1432-0533 (Electronic)&#xD;0001-6322 (Linking)</isbn><accession-num>20012068</accession-num><urls><related-urls><url>;[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 ADDIN EN.CITE <EndNote><Cite ExcludeYear="1"><Author>Padgett</Author><Year>1973</Year><RecNum>6710</RecNum><DisplayText>[10]</DisplayText><record><rec-number>6710</rec-number><foreign-keys><key app="EN" db-id="fzffee0d8fw0xmeet9655992wpfxp9w2rtzf" timestamp="1442358766">6710</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Padgett, B. L.</author><author>Walker, D. L.</author></authors></contributors><titles><title>Prevalence of antibodies in human sera against JC virus, an isolate from a case of progressive multifocal leukoencephalopathy</title><secondary-title>J Infect Dis</secondary-title></titles><periodical><full-title>J Infect Dis</full-title><abbr-1>The Journal of infectious diseases</abbr-1></periodical><pages>467-70</pages><volume>127</volume><number>4</number><keywords><keyword>Adolescent</keyword><keyword>Adult</keyword><keyword>Aged</keyword><keyword>Antibodies, Viral/*analysis</keyword><keyword>Antibody Specificity</keyword><keyword>Brain/microbiology</keyword><keyword>Cells, Cultured</keyword><keyword>Child</keyword><keyword>Child, Preschool</keyword><keyword>Erythrocytes/immunology</keyword><keyword>Female</keyword><keyword>Fluorescent Antibody Technique</keyword><keyword>Hemagglutination Inhibition Tests</keyword><keyword>Hemagglutination Tests</keyword><keyword>Humans</keyword><keyword>Immune Sera</keyword><keyword>Infant</keyword><keyword>Leukoencephalopathy, Progressive Multifocal/*microbiology</keyword><keyword>Male</keyword><keyword>Middle Aged</keyword><keyword>Neutralization Tests</keyword><keyword>Papillomaviridae/*immunology/isolation &amp; purification/pathogenicity</keyword><keyword>*Polyomaviridae</keyword><keyword>Wisconsin</keyword></keywords><dates><year>1973</year><pub-dates><date>Apr</date></pub-dates></dates><isbn>0022-1899 (Print)&#xD;0022-1899 (Linking)</isbn><accession-num>4571704</accession-num><urls><related-urls><url>;[10], we did not detect it in any other sample apart from PT-5 (Supplementary Table 4). ADDIN EN.REFLIST Literature Cited1.Boehme, C.C., et al., Rapid molecular detection of tuberculosis and rifampin resistance. N Engl J Med, 2010. 363(11): p. 1005-15.2.Simner, P.J., et al., Broad-range direct detection and identification of fungi by use of the PLEX-ID PCR-electrospray ionization mass spectrometry (ESI-MS) system. J Clin Microbiol, 2013. 51(6): p. 1699-706.3.Altschul, S.F., et al., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res, 1997. 25(17): p. 3389-402.4.Langmead, B. and S.L. Salzberg, Fast gapped-read alignment with Bowtie 2. Nat Methods, 2012. 9(4): p. 357-9.5.Ferenczy, M.W., et al., Molecular biology, epidemiology, and pathogenesis of progressive multifocal leukoencephalopathy, the JC virus-induced demyelinating disease of the human brain. Clin Microbiol Rev, 2012. 25(3): p. 471-506.6.Khalil, M., et al., Epstein-Barr virus encephalitis presenting with a tumor-like lesion in an immunosuppressed transplant recipient. J Neurovirol, 2008. 14(6): p. 574-8.7.Pardo, C.A., R. Nabbout, and A.S. Galanopoulou, Mechanisms of epileptogenesis in pediatric epileptic syndromes: Rasmussen encephalitis, infantile spasms, and febrile infection-related epilepsy syndrome (FIRES). Neurotherapeutics, 2014. 11(2): p. 297-310.8.Maginnis, M.S. and W.J. Atwood, JC virus: an oncogenic virus in animals and humans? Semin Cancer Biol, 2009. 19(4): p. 261-9.9.Sofroniew, M.V. and H.V. Vinters, Astrocytes: biology and pathology. Acta Neuropathol, 2010. 119(1): p. 7-35.10.Padgett, B.L. and D.L. Walker, Prevalence of antibodies in human sera against JC virus, an isolate from a case of progressive multifocal leukoencephalopathy. J Infect Dis, 1973. 127(4): p. 467-70.11.Morgulis, A., et al., A fast and symmetric DUST implementation to mask low-complexity DNA sequences. J Comput Biol, 2006. 13(5): p. 1028-40.12.Salter, S.J., et al., Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol, 2014. 12(1): p. 87.13.Segata, N., et al., Metagenomic microbial community profiling using unique clade-specific marker genes. Nat Methods, 2012. 9(8): p. 811-4.14.Buchfink, B., C. Xie, and D.H. Huson, Fast and sensitive protein alignment using DIAMOND. Nat Methods, 2015. 12(1): p. 59-60.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. ................
................

In order to avoid copyright disputes, this page is only a partial summary.

Google Online Preview   Download