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Over the past two decades, the diagnosis rate for patients with encephalitis has remained poor despite advances in pathogen-specific testing such as PCR and antigen assays. Metagenomic next-generation sequencing (mNGS) of RNA and DNA extracted from cerebrospinal fluid and brain tissue now offers another strategy for diagnosing neurological infections. Given that mNGS simultaneously assays for a wide range of infectious agents in an unbiased manner, it can identify pathogens that were not part of a neurologist’s initial differential diagnosis either because of the rarity of the infection, because the microorganism has not been previously associated with a clinical phenotype or because it is a newly discovered organism. This Review discusses the technical advantages and pitfalls of cerebrospinal fluid mNGS in the context of patients with neuroinflammatory syndromes, including encephalitis, meningitis and myelitis. We also speculate on how mNGS testing potentially fits into current diagnostic testing algorithms given data on mNGS test performance, cost and turnaround time. Finally, the Review highlights future directions for mNGS technology and other hypothesis-free testing methodologies that are in development., This Review discusses the advantages and pitfalls of metagenomic next-generation sequencing (mNGS) in patients with encephalitis, meningitis and myelitis. The authors outline data on mNGS test performance, cost and turnaround time and highlight future directions for mNGS technology., Key points Meningoencephalitis remains a challenging diagnosis owing to the multitude of possible infectious and autoimmune causes.Meningoencephalitis is associated with a high rate of morbidity and mortality and requires prompt diagnosis and treatment.Metagenomic next-generation sequencing (mNGS) is now a clinically validated test for neuroinfectious diseases that can aid clinicians with a timely diagnosis.mNGS can improve the detection of pathogens that were missed by clinicians or on standard direct testing.mNGS does not perform well when indirect tests are required to make the diagnosis (for example, serology), when infections are compartmentalized and for certain low abundance pathogens.The clinical context of the case is required when interpreting the results of mNGS.

International audience; Varicella vaccine meningitis is an uncommon delayed adverse event of vaccination. Varicella vaccine meningitis has been diagnosed in 12 children, of whom 3 were immunocompromised. We now report two additional cases of vaccine meningitis in twice-immunized immunocompetent children and we perform further testing on a prior third case. We used three methods to diagnose or investigate cases of varicella vaccine meningitis, none of which have been used previously on this disease. These include metagenomic next-generation sequencing and cytokine multiplex profiling of cerebrospinal fluid and immunology exome analysis of white blood cells. In one new case, the diagnosis was confirmed by metagenomic next-generation sequencing of cerebrospinal fluid. Both varicella vaccine virus and human herpesvirus 7 DNA were detected. We performed cytokine multiplex profiling on the cerebrospinal fluid of two cases and found ten elevated biomarkers: interferon gamma, interleukins IL-1RA, IL-6, IL-8, IL-10, IL-17F, chemokines CXCL-9, CXCL-10, CCL-2, and G-CSF. In a second new case, we performed immunology exome sequencing on a panel of 356 genes, but no errors were found. After a review of all 14 cases, we concluded that (i) there is no common explanation for this adverse event, but (ii) ingestion of an oral corticosteroid burst 3–4 weeks before onset of vaccine meningitis may be a risk factor in some cases.

Objectives Tuberculous meningitis(TBM) is a common cause of meningitis in sub-Saharan Africa. CSF PCR with GeneXpert RIF/MTB Ultra is only 70% sensitive for detection of definite/probable TBM. Many infections can mimic TBM. Metagenomic next generation sequencing(mNGS) can detect the whole diversity of infectious microbes, but can be insensitive to TB in CSF. We assessed whether leveraging CSF mNGS to identify infections combined with a machine learning classifier(MLC), based on host transcriptomic data generated by mNGS, could enhance diagnostic accuracy for TBM. Methods Prospectively enrolled 347 HIV-infected Ugandan adults with subacute meningitis: RNA/DNA libraries were made from CSF and deep sequenced. Non-human sequences were interrogated to identify pathogens. A host transcriptomic MLC was developed from human RNA transcripts using 70 cases. The MLC and mNGS reporting thresholds were then tested on 108 blinded cases within the cohort. Results mNGS was 75% concordant(27/36) for detecting TB in definite TBM cases and 59% concordant(30/51) in definite/probable TBM combined. 3 TB and 3 non-TB pathogens were detected in the probable TBM group. In the possible TBM/indeterminant groups, mNGS identified 3 cases of TBM and 17 other pathogens. The combined mNGS and host-MLC displayed 83.3%(5/6) sensitivity, 86.8%(59/68) specificity, with an area under the ROC curve of 0.83(p=0.009). Conclusion mNGS identified an array of infectious TBM mimics, including many treatable and vaccine preventable pathogens. mNGS was 75% concordant with definite TBM. We further enhanced the sensitivity of the CSF mNGS assay by developing the first CSF-based host MLC to discriminate between TBM and its mimics

Background Tuberculous meningitis (TBM) has a high fatality rate, with inadequate diagnostic tests being a major contributor. The rollout of Xpert MTB/Rif and Xpert MTB/RIF Ultra (Xpert Ultra) have improved time-to-diagnosis with sensitivities similar to culture, yet test availability and sensitivity are inadequate. The TB lipoarabinomannan lateral flow assay (AlereLAM) offers ease of use, but its low sensitivity in cerebrospinal fluid (CSF) limits clinical utility for TBM. The Fujifilm SILVAMP TB LAM (FujiLAM) assay has excellent sensitivity in urine, but performance on cerebrospinal fluid is uncertain. Methods We conducted a prospective cohort study at Kiruddu National Referral Hospital in Kampala, Uganda, enrolling patients suspected to have TBM. CSF was tested using AlereLAM, Xpert Ultra, culture, and FujiLAM. Results were compared with 2 reference standards: probable and definite TBM or definite TBM alone by the uniform TBM case definition. Results Of 101 patients enrolled (95/101 HIV-positive), 34 had definite TBM and 24 had probable TBM. FujiLAM sensitivity on CSF was 52% (30/58) for definite or probable TBM compared with 55% (32/58) for Xpert Ultra. AlereLAM had lower sensitivity than FujiLAM in the subgroup of patients tested with both assays (14% [4/28] vs 50% [14/28]; P < .01). FujiLAM specificity was 98% (42/43) for patients without probable or definite TBM. Conclusions FujiLAM showed higher sensitivity than AlereLAM, with sensitivity potentially approaching that of Xpert Ultra. FujiLAM could improve time-to-treatment-initiation, especially in settings where the more technical Xpert Ultra system might not be feasible. Large confirmatory studies are needed.

Background: Neurosyphilis (NS) presents with a variety of clinical syndromes that can be attributed to other aetiologies due to difficulties in its diagnosis. We reviewed all cases of NS from the “Top End” of the Australian Northern Territory over a ten-year period to assess incidence, clinical and laboratory manifestations. Methods: Patient data (2007–2016) were extracted from hospital records, centralised laboratory data and Northern Territory Centre for Disease Control records. Clinical records of patients with clinically suspected NS were reviewed. A diagnosis of NS was made based on the 2014 US CDC criteria. Results were also recategorized based on the 2018 US CDC criteria. Results: The population of the “Top End” is 185,570, of whom 26.2% are Indigenous. A positive TPPA was recorded in 3126 individuals. A total of 75 (2.4%) of TPPA positive patients had a lumbar puncture (LP), of whom 25 (35%) were diagnosed with NS (9 definite, 16 probable). Dementia was the most common manifestation (58.3%), followed by epilepsy (16.7%), psychosis (12.5%), tabes dorsalis (12.5%) and meningovascular syphilis (8.3%). 63% of probable NS cases were not treated appropriately due to a negative CSF VDRL. Despite increased specificity of the 2018 US CDC criteria, 70% of patient in the probable NS group were not treated appropriately. The overall annual incidence [95%CI] of NS was 2.47[1.28–4.31] per 100 000py in the Indigenous population and 0.95[0.50–1.62] in the non-Indigenous population (rate ratio = 2.60 [1.19–5.70];p = 0.017). Conclusion: Neurosyphilis is frequently reported in the NT, particularly in Indigenous populations. Disturbingly, 60% of probable neurosyphilis patients based on the 2014 criteria, and 70% based on the 2018 criteria with were not treated appropriately. It is critical that clinicians should be aware of the diagnosis of NS and treat patients appropriately.

Author(s): Beck, Erin S; Ramachandran, Prashanth S; Khan, Lillian M; Sample, Hannah A; Zorn, Kelsey C; O'Connell, Elise M; Nash, Theodore; Reich, Daniel S; Venkatesan, Arun; DeRisi, Joseph L; Nath, Avindra; Wilson, Michael R

We aimed to identify the contribution of central nervous system (CNS) viral coinfection to illness in African children with retinopathy-negative or retinopathy-positive cerebral malaria (CM). We collected cerebrospinal fluid (CSF) from 272 children with retinopathy-negative or retinopathy-positive CM and selected CSF from 111 of these children (38 retinopathy positive, 71 retinopathy negative, 2 retinopathy unknown) for analysis by metagenomic next-generation sequencing. We found CSF viral coinfections in 7/38 (18.4%) retinopathy-positive children and in 18/71 (25.4%) retinopathy-negative children. Excluding HIV-1, human herpesviruses (HHV) represented 61% of viruses identified. Excluding HIV-1, CNS viral coinfection was equally likely in children who were retinopathy positive and retinopathy negative (P = 0.1431). Neither mortality nor neurological morbidity was associated with the presence of virus (odds ratio [OR] = 0.276, 95% CI: 0.056-1.363). Retinopathy-negative children with a higher temperature, lower white blood cell count, or being dehydrated were more likely to have viral coinfection. Level of consciousness at admission was not associated with CNS viral coinfection in retinopathy-negative children. Viral CNS coinfection is unlikely to contribute to coma in children with CM. The herpesviruses other than herpes simplex virus may represent incidental bystanders in CM, reactivating during acute malaria infection.

This case report presents the clinical record of a 37-year-old man who presented with a dense right hemiplegia, found to be caused by a left medial medullary stroke. The cause of the stroke was unclear, and bacterial endocarditis was initially suspected. However, he was ultimately found to have neurosyphilis on a background of undiagnosed human immunodeficiency virus and was treated with benzylpenicillin. This case report reviews the diagnosis of neurosyphilis and highlights the importance of considering neurosyphilis as a rare but important cause of stroke, especially given the increasing prevalence of syphilis in Australia.

OBJECTIVE: In 2016, Catalonia experienced a pediatric brainstem encephalitis outbreak caused by enterovirus A71 (EV-A71). Conventional testing identified EV in the periphery but rarely in CSF. Metagenomic next-generation sequencing (mNGS) and CSF pan-viral serology (VirScan) were deployed to enhance viral detection and characterization. METHODS: RNA was extracted from the CSF (n = 20), plasma (n = 9), stool (n = 15), and nasopharyngeal samples (n = 16) from 10 children with brainstem encephalitis and 10 children with meningitis or encephalitis. Pathogens were identified using mNGS. Available CSF from cases (n = 12) and pediatric other neurologic disease controls (n = 54) were analyzed with VirScan with a subset (n = 9 and n = 50) validated by ELISA. RESULTS: mNGS detected EV in all samples positive by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (n = 25). In qRT-PCR-negative samples (n = 35), mNGS found virus in 23% (n = 8, 3 CSF samples). Overall, mNGS enhanced EV detection from 42% (25/60) to 57% (33/60) (p-value = 0.013). VirScan and ELISA increased detection to 92% (11/12) compared with 46% (4/12) for CSF mNGS and qRT-PCR (p-value = 0.023). Phylogenetic analysis confirmed the EV-A71 strain clustered with a neurovirulent German EV-A71. A single amino acid substitution (S241P) in the EVA71 VP1 protein was exclusive to the CNS in one subject. CONCLUSION: mNGS with VirScan significantly increased the CNS detection of EVs relative to qRT-PCR, and the latter generated an antigenic profile of the acute EV-A71 immune response. Genomic analysis confirmed the close relation of the outbreak EV-A71 and neuroinvasive German EV-A71. A S241P substitution in VP1 was found exclusively in the CSF. Grants supporting this project include the National Multiple Sclerosis Society and the American Academy of Neurology award FAN-1608-25607 (R.D.S.), Clinical Research Training Scholarship P0534134 (P.S.R.), Sandler and William K. Bowes Jr Foundations (M.R.W., J.L.D., L.M.K., H.A.S., K.C.Z.), Rachleff Family Foundation (M.R.W.), and NINDS of the NIH under award K08NS096117 (M.R.W.) and F31NS113432 (K.E.L.). This study was partially supported by a grant from the Spanish National Health Institute [grant number PI15CIII-00020] and the European Regional Development Fund (FEDER funds). UCSF Biomedical Sciences Program (I.A.H., K.E.L.), UCSF Medical Scientist Training Program (K.E.L.), and the Chan Zuckerberg Biohub (J.E.P., W.W., C.K.C., J.L.D., E.D.C.) also supported this project. Sí

Neuroinfectious diseases continue to cause morbidity and mortality worldwide, with many emerging or reemerging infections resulting in neurologic sequelae. Careful clinical evaluation coupled with appropriate laboratory investigations still forms the bedrock for making the correct etiologic diagnosis and implementing appropriate management. The treating physician needs to understand the individual test characteristics of each of the many conventional candidate-based diagnostics: culture, pathogen-specific polymerase chain reaction, antigen, antibody tests, used to diagnose the whole array of neuroinvasive infections. In addition, there is a growing need for more comprehensive, agnostic testing modalities that can identify a diversity of infections with a single assay.

Highlights: Consider botulism in a patient with weakness, ptosis, internal and external ophthalmoplegia and autonomic features. In contrast to acute neuropathies (e.g. Miller Fisher syndrome), the reflexes are usually preserved. Toxin serotype may predict clinical course and severity. An anti-toxin is available and should be given early and rigorous case follow-up is vital in food borne outbreaks. With good supportive care, the prognosis is excellent but recovery is often protracted over many months.

A 40-year-old Somali woman presented features of a right-sided cavernous sinus syndrome which was confirmed with neuroimaging. Although initial investigations were equivocal for an infectious etiology, subsequent investigations led to a diagnosis of tuberculosis as the cause for right-sided cavernous sinus syndrome. This case illustrates that, although the incidence of tuberculosis cavernous sinus syndrome is reportedly low, patients originating from tuberculosis endemic regions warrant scrupulous investigations in order not to miss the diagnosis and effect appropriate treatment.

Objectives Neurosyphilis (NS) can present in a variety of clinical syndromes and may be attributed to other aetiologies due to difficulties in its diagnosis. We reviewed all cases of NS from the ‘Top End’ of the Northern Territory over a ten-year period to assess incidence, clinical and laboratory manifestations. Methods Patient data (2007–2016) was extracted from hospital records, centralised laboratory data and the Centre for Disease Control (CDC). The clinical records of patients with clinically suspected NS were reviewed. A diagnosis of confirmed NS was made in those patients with a positive serum Treponema pallidum particle agglutination assay (TPPA) and CSF Venereal Disease Research Laboratory (VDRL) test. Probable NS was made in those with a positive serum TPPA and abnormal CSF. Results The population of the ‘Top End’ is 170 000, of whom 27% are indigenous. A positive TPPA was recorded in 3112 individuals. A total of 75 (2.4%) of TPPA positive patients had a lumbar puncture (LP), of whom 28 (37%) were diagnosed with NS (9 confirmed, 19 probable) and a further two patients had possible NS. Dementia was the most common manifestation (18/29) followed by meningovascular (3), psychosis (4), tabes dorsalis (3) and epilepsy (2). Nine (30%) were not treated for NS due to a negative CSF VDRL despite meeting criteria. The overall incidence (95% CI) of NS was 33 (18–54) per 100 000 person years in the indigenous population and 12 (7–20) in the non-indigenous population (rate ratio=2.7 (1.3–5.5); p=0.0064). Conclusions Syphilis is common in the NT and NS is frequently reported, particularly in indigenous populations. Disturbingly, nearly a third of patients with NS were not treated appropriately because of over-reliance on CSF VDRL positivity. We propose a systematic approach to the diagnosis of NS, which will avoid misdiagnosis.