Biology subjects

DeRisi, J. L.

Publications and source records attributed to DeRisi, J. L..

At least 37 records · Page 2Linked to original sources

bioRxiv2020

IDseq - An Open Source Cloud-based Pipeline and Analysis Service for Metagenomic Pathogen Detection and Monitoring

BackgroundMetagenomic next generation sequencing (mNGS) has enabled the rapid, unbiased detection and identification of microbes without pathogen-specific reagents, culturing, or a priori knowledge of the microbial landscape. mNGS data analysis requires a series of computationally intensive processing steps to accurately determine the microbial composition of a sample. Existing mNGS data analysis tools typically require bioinformatics expertise and access to local server-class hardware resources. For many research laboratories, this presents an obstacle, especially in resource limited environments. FindingsWe present IDseq, an open source cloud-based metagenomics pipeline and service for global pathogen detection and monitoring (https://idseq.net). The IDseq Portal accepts raw mNGS data, performs host and quality filtration steps, then executes an assembly-based alignment pipeline which results in the assignment of reads and contigs to taxonomic categories. The taxonomic relative abundances are reported and visualized in an easy-to-use web application to facilitate data interpretation and hypothesis generation. Furthermore, IDseq supports environmental background model generation and automatic internal spike-in control recognition, providing statistics which are critical for data interpretation. IDseq was designed with the specific intent of detecting novel pathogens. Here, we benchmark novel virus detection capability using both synthetically evolved viral sequences, and real-world samples, including IDseq analysis of a nasopharyngeal swab sample acquired and processed locally in Cambodia from a tourist from Wuhan, China, infected with the recently emergent SARS-CoV-2. ConclusionThe IDseq Portal reduces the barrier to entry for mNGS data analysis and enables bench scientists, clinicians, and bioinformaticians to gain insight from mNGS datasets for both known and novel pathogens.

bioinformatics↗

bioRxiv2020

Identification of novel, clinically correlated autoantigens in the monogenic autoimmune syndrome APS1 by PhIP-Seq

The identification of autoantigens remains a critical challenge for understanding and treating autoimmune diseases. Autoimmune polyendocrine syndrome type 1 (APS1), a rare monogenic form of autoimmunity, presents as widespread autoimmunity with T and B cell responses to multiple organs. Importantly, autoantibody discovery in APS1 can illuminate fundamental disease pathogenesis, and many of the antigens found in APS1 extend to common autoimmune diseases. Here, we performed proteome-wide programmable phage-display (PhIP-Seq) on sera from an APS1 cohort and discovered multiple common antibody targets. These novel autoantigens exhibit tissue-restricted expression, including expression in enteroendocrine cells and dental enamel. Using detailed clinical phenotyping, we find novel associations between autoantibodies and organ-restricted autoimmunity, including between anti-KHDC3L autoantibodies and premature ovarian insufficiency, and between anti-RFX6 autoantibodies and diarrheal-type intestinal dysfunction. Our study highlights the utility of PhIP-Seq for interrogating antigenic repertoires in human autoimmunity and the importance of antigen discovery for improved understanding of disease mechanisms.

immunology↗

bioRxiv2019

An exploration of ambigrammatic sequences in narnaviruses

Narnaviruses have been described as positive-sense RNA viruses with a remarkably simple genome of [~] 3 kb, encoding only a highly conserved RNA-dependent RNA polymerase (RdRp). Many narnaviruses, however, are ambigrammatic and harbour an additional uninterrupted open reading frame (ORF) covering almost the entire length of the reverse complement strand. No function has been described for this ORF, yet the absence of stops is conserved across diverse narnaviruses, and in every case the codons in the reverse ORF and the RdRp are aligned. The > 3 kb ORF overlap on opposite strands, unprecedented among RNA viruses, motivates an exploration of the constraints imposed or alleviated by the codon alignment. Here, we show that only when the codon frames are aligned can all stop codons be eliminated from the reverse strand by synonymous single-nucleotide substitutions in the RdRp gene, suggesting a mechanism for de novo gene creation within a strongly conserved amino-acid sequence. It will be fascinating to explore what implications this coding strategy has for other aspects of narnavirus biology. Beyond narnaviruses, our rapidly expanding catalogue of viral diversity may yet reveal additional examples of this broadly-extensible principle for ambigrammatic-sequence development.

genomics↗

bioRxiv2018

Exploration of Anti-Yo and Anti-Hu paraneoplastic neurological disorders by PhIP-Seq reveals a highly restricted pattern of antibody epitopes

Paraneoplastic neurological disorders (PNDs) are immune-mediated diseases of the nervous system understood to manifest as part of a misdirected anti-tumor immune response. Identifying PND-associated autoantibodies and their cognate antigens can assist with proper diagnosis and treatment while also enhancing our understanding of tumor-associated immune processes, triggers for autoimmune disease, and the functional significance of onconeuronal proteins. Here, we employed an enhanced version of phage display immunoprecipitation and sequencing (PhIP-Seq) leveraging a library of over 731,000 unique phage clones tiling across the entire human proteome to detect autoantibodies and create high-resolution epitope profiles in serum and CSF samples from patients suffering from two common PNDs, the anti-Yo (n = 36 patients) and anti-Hu syndromes (n = 44 patients). All patient samples positive for anti-Yo antibody by a validated clinical assay yielded polyspecific enrichment of phage presenting peptides from the canonical anti-Yo (CDR2 and CDR2L) antigens, while 38% of anti-Hu patients (17/44) had a serum and/or CSF sample that significantly enriched peptides deriving from the ELAVL family of proteins, the anti-Hu autoantigenic target. The anti-Hu antibodies showed a remarkably convergent antigenic signature across 15/17 patients corresponding to residues surrounding and including the degenerate motif, RLDxLL, shared by ELAVL2, 3 and 4. Lastly, PhIP-Seq identified several known and novel autoantigens in these same patient samples, representing potential biomarkers that could aid in the diagnosis and prognosis of PND and cancer.

immunology↗

bioRxiv2018

Miniaturization and optimization of 384-well compatible metagenomic sequencing library preparation

Preparation of high-quality sequencing libraries is a costly and time-consuming component of metagenomic next generation sequencing (mNGS). While the overall cost of sequencing has dropped significantly over recent years, the reagents needed to prepare sequencing samples are likely to become the dominant expense in the process. Furthermore, libraries prepared by hand are subject to human variability and needless waste due to limitations of manual pipetting volumes. Reduction of reaction volumes, combined with sub-microliter automated dispensing of reagents without consumable pipette tips, has the potential to provide significant advantages. Here, we describe the integration of several instruments, including the Labcyte Echo 525 acoustic liquid handler and the iSeq and NovaSeq Illumina sequencing platforms, to miniaturize and automate mNGS library preparation, significantly reducing the cost and the time required to prepare samples. Through the use of External RNA Controls Consortium (ERCC) spike-in RNAs, we demonstrated the fidelity of the miniaturized preparation to be equivalent to full volume reactions. Furthermore, detection of viral and microbial species from cell culture and patient samples was also maintained in the miniaturized libraries. For 384-well mNGS library preparations, we achieved a savings of over 80% in materials and reagents alone, and reduced preparation time by 90% compared to manual approaches, without compromising quality or representation within the library.

molecular biology↗

bioRxiv2018

FLASH: A next-generation CRISPR diagnostic for multiplexed detection of antimicrobial resistance sequences

The growing prevalence of deadly microbes with resistance to previously life-saving drug therapies is a dire threat to human health. Detection of low abundance pathogen sequences remains a challenge for metagenomic Next Generation Sequencing (NGS). We introduce FLASH (Finding Low Abundance Sequences by Hybridization), a next-generation CRISPR/Cas9 diagnostic method that takes advantage of the efficiency, specificity and flexibility of Cas9 to enrich for a programmed set of sequences. FLASH-NGS achieves up to 5 orders of magnitude of enrichment and sub-attomolar gene detection with minimal background. We provide an open-source software tool (FLASHit) for guide RNA design. Here we applied it to detection of antimicrobial resistance genes in respiratory fluid and dried blood spots, but FLASH-NGS is applicable to all areas that rely on multiplex PCR.

microbiology↗

bioRxiv2018

Pediatric Brainstem Encephalitis Outbreak Investigation with Metagenomic Next-Generation Sequencing

In 2016, Catalonia experienced a pediatric brainstem encephalitis outbreak caused by enterovirus A71 (EV-A71). Conventional testing identified EV in peripheral body sites, but EV was rarely identified in cerebrospinal fluid (CSF). RNA was extracted from CSF (n=20), plasma (n=9), stool (n=15) and nasopharyngeal samples (n=16) from 10 children with brainstem encephalitis or encephalomyelitis and 10 contemporaneous pediatric controls with presumed viral meningitis or encephalitis. Unbiased complementary DNA libraries were sequenced, and microbial pathogens were identified using a custom bioinformatics pipeline. Full-length virus genomes were assembled for phylogenetic analyses. Metagenomic next-generation sequencing (mNGS) was concordant with qRT-PCR for all samples positive by PCR (n=25). In virus-negative samples (n=35), mNGS detected virus in 28.6% (n=10), including 5 CSF samples. mNGS co-detected EV-A71 and another EV in 5 patients. Overall, mNGS increased the proportion of EV-positive samples from 42% (25/60) to 57% (34/60) (McNemars test; p-value = 0.0077). For CSF, mNGS doubled the number of pathogen-positive samples (McNemars test; p-value = 0.074). Using phylogenetic analysis, the outbreak EV-A71 clustered with a neuroinvasive German EV-A71 isolate. Brainstem encephalitis specific, non-synonymous EV-A71 single nucleotide variants were not identified. mNGS demonstrated 100% concordance with clinical qRT-PCR of EV-related brainstem encephalitis and significantly increased the detection of enteroviruses. Our findings increase the probability that neurologic complications observed were virus-induced rather than para-infectious. A comprehensive genomic analysis confirmed that the EV-A71 outbreak strain was closely related to a neuroinvasive German EV-A71 isolate. There were no clear-cut viral genomic differences that discriminated between patients with differing neurologic phenotypes.

microbiology↗

bioRxiv2018

The Plasmodium falciparum cytoplasmic translation apparatus: a promising therapeutic target not yet exploited by clinically approved antimalarials.

The continued specter of resistance to existing antimalarials necessitates the pursuit of novel targets and mechanisms of action for drug development. One class of promising targets consists of the 80S ribosome and its associated components comprising the parasite translational apparatus. Development of translation-targeting therapeutics requires a greater understanding of protein synthesis and its regulation in the malaria parasite. Research in this area has been limited by the lack of appropriate experimental methods, particularly a direct measure of parasite translation. We have recently developed and optimized the PfIVT assay, an in vitro method directly measuring translation in whole-cell extracts from the malaria parasite Plasmodium falciparum.Here, we present an extensive pharmacologic assessment of the PfIVT assay using a wide range of known inhibitors, demonstrating its utility for studying activity of both ribosomal and non-ribosomal elements directly involved in translation. We further demonstrate the superiority of this assay over a historically utilized indirect measure of translation, S35-radiolabel incorporation. Additionally, we utilize the PfIVT assay to investigate a panel of clinically approved antimalarial drugs, many with unknown or unclear mechanisms of action, and show that none inhibit translation, reaffirming Plasmodium translation to be a viable alternative drug target. Within this set, we unambiguously find that mefloquine lacks translation inhibition activity, despite having been recently mischaracterized as a ribosomal inhibitor. This work exploits a direct and reproducible assay for measuring P. falciparum translation, demonstrating its value in the continued study of protein synthesis in malaria and its inhibition as a drug target.\n\nAuthor summaryNovel antimalarial drugs are required to combat rising resistance to current therapies. The protein synthesis machinery of the malaria parasite Plasmodium falciparum is a promising unexploited target for antimalarial development, but its study has been hindered by use of indirect experimental methods which often produce misleading and inaccurate results. We have recently developed a direct method to investigate malaria protein synthesis utilizing whole-parasite extracts. In this work, we present an extensive characterization of the assay, using a panel of pharmacologic inhibitors with known mechanisms of action. We demonstrate the specificity of the assay in various stages of protein synthesis, as well as its improved accuracy and sensitivity in comparison to an indirect measure that has been the previous standard for the field. We further demonstrate that no current clinically available antimalarial drugs inhibit protein synthesis, emphasizing its potential as a target for drugs that will overcome existing resistance. Importantly, among the antimalarials tested was mefloquine, a widely used antimalarial that has recently been mischaracterized as an inhibitor protein synthesis. Our finding that mefloquine does not inhibit protein synthesis emphasizes the importance of using direct functional measurements when determining drug targets.

molecular biology↗

bioRxiv2018

Etiology of fever in Ugandan children: identification of microbial pathogens using metagenomic next-generation sequencing and IDseq, a platform for unbiased metagenomic analysis

BackgroundFebrile illness is a major burden in African children, and non-malarial causes of fever are uncertain. We built and employed IDseq, a cloud-based, open access, bioinformatics platform and service to identify microbes from metagenomic next-generation sequencing of tissue samples. In this pilot study, we evaluated blood, nasopharyngeal, and stool specimens from 94 children (aged 2-54 months) with febrile illness admitted to Tororo District Hospital, Uganda.\n\nResultsThe most common pathogens identified were Plasmodium falciparum (51.1% of samples) and parvovirus B19 (4.4%) from blood; human rhinoviruses A and C (40%), respiratory syncytial virus (10%), and human herpesvirus 5 (10%) from nasopharyngeal swabs; and rotavirus A (50% of those with diarrhea) from stool. Among other potential pathogens, we identified one novel orthobunyavirus, tentatively named Nyangole virus, from the blood of a child diagnosed with malaria and pneumonia, and Bwamba orthobunyavirus in the nasopharynx of a child with rash and sepsis. We also identified two novel human rhinovirus C species.\n\nConclusionsThis exploratory pilot study demonstrates the utility of mNGS and the IDseq platform for defining the molecular landscape of febrile infectious diseases in resource limited areas. These methods, supported by a robust data analysis and sharing platform, offer a new tool for the surveillance, diagnosis, and ultimately treatment and prevention of infectious diseases.

genomics↗

bioRxiv2018

A Metagenomic Comparison of Tracheal Aspirate and Mini-Bronchial Alveolar Lavage for Assessment of Respiratory Microbiota

Lower respiratory tract infections (LRTI) lead to more deaths each year than any other infectious disease category. Despite this, etiologic LRTI pathogens are infrequently identified due to limitations of existing microbiologic tests. In critically ill patients, non-infectious inflammatory syndromes resembling LRTI further complicate diagnosis. To address the need for improved LRTI diagnostics, we performed metagenomic next-generation sequencing (mNGS) on tracheal aspirates from 92 adults with acute respiratory failure and simultaneously assessed pathogens, the airway microbiome and the host transcriptome. To differentiate pathogens from respiratory commensals, we developed rules-based and logistic regression models (RBM, LRM) in a derivation cohort of 20 patients with LRTI or non-infectious acute respiratory illnesses. When tested in an independent validation cohort of 24 patients, both models achieved accuracies of 95.5%. We next developed pathogen, microbiome diversity, and host gene expression metrics to identify LRTI-positive patients and differentiate them from critically ill controls with non-infectious acute respiratory illnesses. When tested in the validation cohort, the pathogen metric performed with an AUC of 0.96 (95% CI = 0.86 - 1.00), the diversity metric with an AUC of 0.80 (95% CI = 0.63 - 0.98), and the host transcriptional classifier with an AUC of 0.88 (95% CI = 0.75 - 1.00). Combining these achieved a negative predictive value of 100%. This study suggests that a single streamlined protocol offering an integrated genomic portrait of pathogen, microbiome and host transcriptome may hold promise as a novel tool for LRTI diagnosis.\n\nSIGNIFICANCE STATEMENTLower respiratory tract infections (LRTI) are the leading cause of infectious disease-related death worldwide yet remain challenging to diagnose because of limitations in existing microbiologic tests. In critically ill patients, non-infectious respiratory syndromes that resemble LRTI further complicate diagnosis and confound targeted treatment. To address this, we developed a novel metagenomic sequencing-based approach that simultaneously interrogates three core elements of acute airway infections: the pathogen, airway microbiome and host response. We studied this approach in a prospective cohort of critically ill patients with acute respiratory failure and found that combining pathogen, microbiome and host gene expression metrics achieved accurate LRTI diagnosis and identified etiologic pathogens in patients with clinically identified infections but otherwise negative testing.

microbiology↗

bioRxiv2018

Integrating Host Response and Unbiased Microbe Detection for Lower Respiratory Tract Infection Diagnosis in Critically Ill Adults

Lower respiratory tract infections (LRTI) lead to more deaths each year than any other infectious disease category(1). Despite this, etiologic LRTI pathogens are infrequently identified due to limitations of existing microbiologic tests(2). In critically ill patients, non-infectious inflammatory syndromes resembling LRTI further complicate diagnosis. To address the need for improved LRTI diagnostics, we performed metagenomic next-generation sequencing (mNGS) on tracheal aspirates from 92 adults with acute respiratory failure and simultaneously assessed pathogens, the lung microbiome and the host transcriptome. To differentiate pathogens from respiratory commensals, we developed rules-based and logistic regression models (RBM, LRM) in a derivation cohort of 20 patients with LRTI or non-infectious acute respiratory illnesses. When tested in an independent validation cohort of 24 patients, both models achieved accuracies of 95.5%. We next developed pathogen, microbiome diversity, and host gene expression metrics to identify LRTI-positive patients and differentiate them from critically ill controls with non-infectious acute respiratory illnesses. When tested in the validation cohort, the pathogen metric performed with an AUC of 0.96 (95% CI = 0.86 - 1.00), the diversity metric with an AUC of 0.80 (95% CI = 0.63 - 0.98), and the host transcriptional classifier with an AUC of 0.91 (95% CI = 0.80 - 1.00). Combining all three achieved an AUC of 0.99 (95% CI = 0.97 - 1.00) and negative predictive value of 100%. This study suggests that a single streamlined protocol offering an integrated genomic portrait of pathogen, microbiome and host transcriptome may hold promise as a novel tool for LRTI diagnosis.\n\nSIGNIFICANCE STATEMENTLower respiratory tract infections (LRTI) are the leading cause of infectious disease-related death worldwide yet remain challenging to diagnose because of limitations in existing microbiologic tests. In critically ill patients, non-infectious respiratory syndromes that resemble LRTI further complicate diagnosis and confound targeted treatment. To address this, we developed a novel metagenomic sequencing-based approach that simultaneously interrogates three core elements of acute airway infections: the pathogen, lung microbiome and host response. We studied this approach in a prospective cohort of critically ill patients with acute respiratory failure and found that combining pathogen, microbiome and host gene expression metrics achieved accurate LRTI diagnosis and identified etiologic pathogens in patients with clinically identified infections but otherwise negative testing.\n\nFundingNHLBI K12HL119997 (Langelier C), NHLBI K23HL123778 (Christensen S), NIAID P01AI091575 and the Chan Zuckerberg Biohub (DeRisi JL), NHLBI K23 HL136844 (Moazed F), NHLBI R01HL110969, K24HL133390, R35HL140026 (Calfee C), Gladstone Institutes (Pollard KS).

genomics↗

bioRxiv2018

Repurposing the quinoline antibiotic nitroxoline to treat infections caused by the brain-eating amoeba Balamuthia mandrillaris

Balamuthia mandrillaris is a pathogenic free-living amoeba that causes a rare but almost always fatal infection of the central nervous system called granulomatous amoebic encephalitis (GAE). Two distinct forms of B. mandrillaris - a proliferative trophozoite form and a non-proliferative cyst form, which is highly resistant to harsh physical and chemical conditions - have been isolated from environmental samples worldwide and are both observed in infected tissue. Patients suffering from GAE are typically treated with aggressive and prolonged multi-drug regimens often including the antimicrobial agents miltefosine and pentamidine isethionate. However, survival rates remain low and studies evaluating the susceptibility of B. mandrillaris to these compounds and other potential therapeutics are limited. To address the need for more effective treatments, we screened 2,177 clinically-approved compounds for in vitro activity against B. mandrillaris. The quinoline antibiotic nitroxoline, which has safely been used in humans to treat urinary tract infections, was identified as a lead compound. We show that nitroxoline inhibits both trophozoites and cysts at low micromolar concentrations, which are within a physiologically relevant range. We compare the in vitro efficacy of nitroxoline to drugs currently used in the standard of care for GAE and find that nitroxoline is the most potent and selective inhibitor of B. mandrillaris tested. Furthermore, we demonstrate that nitroxoline prevents B. mandrillaris-mediated destruction of host cells in cultured fibroblast and primary brain explant models also at physiologically relevant concentrations. Together, our findings indicate that nitroxoline is a promising candidate for repurposing as a novel treatment of B. mandrillaris infections.\n\nImportanceBalamuthia mandrillaris is responsible for hundreds of reported cases of amoebic encephalitis, the majority of which have been fatal. Despite being an exceptionally deadly pathogen, B. mandrillaris is understudied, leaving many open questions regarding epidemiology, diagnosis, and treatment. Due to the lack of effective drugs to fight B. mandrillaris infections, mortality rates remain high even for patients receiving intensive care. This study addresses the need for new anti-amoebic drugs using a high-throughput screening approach to identify novel B. mandrillaris inhibitors. The most promising candidate identified was the quinoline antibiotic nitroxoline, which has a long history of safe use in humans. We show that nitroxoline kills B. mandrillaris at physiologically relevant concentrations and exhibits greater potency and selectivity than drugs commonly used in the current standard of care. The findings we present demonstrate the potential of nitroxoline to be an important new tool in the treatment of life threatening B. mandrillaris infections.

pharmacology and toxicology↗

bioRxiv2018

Evidence for Alternative Complement Cascade Activation in Primary CNS Vasculitis

The central nervous system (CNS) has a dedicated network of blood vessels to support the physiological activity of the brain, spinal cord and meninges. Consequently, inflammation of CNS vasculature can have devastating effects on neurological function. A lack of understanding regarding the molecular pathology of CNS vasculitis impedes the development of better diagnostics and effective therapies. Here, we analyze the proteome of cerebrospinal fluid from patients with biopsy-confirmed Primary Angiitis of the Central Nervous System (PACNS) relative to non-inflammatory control patients and patients with Reversible Cerebral Vasoconstrictive Syndrome (RCVS), a syndrome that clinically mimics PACNS in several aspects. In PACNS, we find significant elevation of apolipoproteins, immunoglobulins and complement cascade components. Notably, we find a bias towards activation of the alternative complement pathway with elevated levels of the terminal cascade component, complement C5. Given the recent treatment successes of Anti-Neutrophil Cytoplasmic Antibody (ANCA) vasculitis with the C5 receptor inhibitor, CCX168 (Avacopan), our results suggest that complement C5 inhibitors may also prove useful as therapeutic interventions for PACNS.

immunology↗

bioRxiv2018

Metagenomic next-generation sequencing reveals Miamiensis avidus (Ciliophora: Scuticociliatida) in the 2017 epizootic of leopard sharks (Triakis semifasciata) in San Francisco Bay, California

During March to August of 2017, hundreds of leopard sharks (Triakis semifasciata) stranded and died on the shores of San Francisco Bay, California, USA. Similar mass stranding events occurred in 1967 and 2011, yet analysis of these epizootics was incomplete and no etiology was confirmed. Our investigation of the most recent epizootic revealed severe meningoencephalitis in stranded sharks, raising suspicion for infection. On this basis, we pursued a strategy for unbiased pathogen detection using metagenomic next-generation sequencing followed by orthogonal validation and further screening. We show that the ciliated protozoan pathogen, Miamiensis avidus, was present in the central nervous system of leopard (n=12) and other shark species (n=2) that stranded in San Francisco Bay, but absent in leopard sharks caught elsewhere. Whereas this protozoan has previously been implicated in devastating outbreaks in teleost marine fish, our findings represent the first report of a ciliated protozoan infection in wild elasmobranchs. This discovery highlights the benefits of adopting unbiased metagenomic sequencing in the study of wildlife health and disease.

microbiology↗

bioRxiv2018

Pulmonary Metagenomic Sequencing Suggests Missed Infections in Immunocompromised Children

RATIONALEDespite improved diagnostics, pulmonary pathogens in immunocompromised children frequently evade detection, leading to significant morbidity and mortality.\n\nOBJECTIVESTo develop a highly sensitive metagenomic next generation sequencing (mNGS) assay capable of evaluating the pulmonary microbiome and identifying diverse pathogens in the lungs of immunocompromised children.\n\nMETHODSWe collected 41 lower respiratory specimens from 34 immunocompromised children undergoing evaluation for pulmonary disease at 3 childrens hospitals from 2014-2016. Samples underwent mechanical homogenization, paired RNA/DNA extraction, and metagenomic sequencing. Sequencing reads were aligned to the NCBI nucleotide reference database to determine taxonomic identities. Statistical outliers were determined based on abundance within each sample and relative to other samples in the cohort.\n\nMEASUREMENTS & MAIN RESULTSWe identified a rich cross-domain pulmonary microbiome containing bacteria, fungi, RNA viruses, and DNA viruses in each patient. Potentially pathogenic bacteria were ubiquitous among samples but could be distinguished as possible causes of disease by parsing for outlier organisms. Samples with bacterial outliers had significantly depressed alpha-diversity (median 0.58, IQR 0.33-0.62 vs. median 0.94, IQR 0.93-0.95, p<0.001). Potential pathogens were detected in half of samples previously negative by clinical diagnostics, demonstrating increased sensitivity for missed pulmonary pathogens (p<0.001).\n\nCONCLUSIONSAn optimized mNGS assay for pulmonary microbes demonstrates significant inoculation of the lower airways of immunocompromised children with diverse bacteria, fungi, and viruses. Potential pathogens can be identified based on absolute and relative abundance. Ongoing investigation is needed to determine the pathogenic significance of outlier microbes in the lungs of immunocompromised children with pulmonary disease.

microbiology↗

bioRxiv2017

Metagenomics for chronic meningitis: clarifying interpretation and diagnosis

ImportanceIdentifying infectious causes of subacute and chronic meningitis can be challenging. Enhanced, unbiased diagnostic approaches are needed.\n\nObjectiveTo present a case series of patients with diagnostically challenging subacute and chronic meningitis in whom metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF), supported by a statistical framework generated from mNGS sequencing of non-infectious patients and environmental controls, identified a pathogen.\n\nDesignCase series. Using mNGS data from the CSF of 94 non-infectious neuroinflammatory cases and 24 water and reagent controls, we developed and implemented a weighted scoring metric based on z-scores at the species and genus level for both nucleotide and protein databases to prioritize and rank mNGS results. We performed mNGS on total RNA extracted from CSF of patients with subacute or chronic meningitis and highlight seven cases representing a diverse array of pathogens.\n\nSettingA multi-center study of mNGS pathogen discovery in patients with suspected neuroinflammatory conditions.\n\nParticipantsPatients with diagnostically challenging subacute or chronic meningitis enrolled in a research study of mNGS performed on CSF.\n\nInterventionmNGS was performed on total RNA extracted from CSF (0.25-0.5 mL). A weighted z-score was used to filter out environmental contaminants and facilitate efficient data triage and analysis.\n\nMain Outcomes1) Pathogens identified by mNGS and 2) ability of a statistical model to prioritize, rank, and simplify mNGS results.\n\nResultsmNGS identified parasitic worms, fungi and viruses in seven subjects: Taenia solium (n=2), Cryptococcus neoformans, human immunodeficiency virus-1, Aspergillus oryzae, Histoplasma capsulatum, and Candida dubliniensis. Evaluating mNGS data with a weighted z-score based scoring algorithm effectively separated bona fide pathogen sequences from spurious environmental sequences.\n\nConclusions and RelevancemNGS of CSF identified a diversity of microbial pathogens in patients with diagnostically challenging subacute or chronic meningitis, including a case of subarachnoid neurocysticercosis that defied diagnosis for one year, the first case of CNS vasculitis caused by Aspergillus oryzae, and the fourth reported case of Candida dubliniensis meningitis. Filtering metagenomic data with a scoring algorithm greatly clarified data interpretation and highlights the difficulties attributing biological significance to organisms that may be present in control samples used for metagenomic sequencing studies.\n\nKey PointsQuestion: How can metagenomic next-generation sequencing of cerebrospinal fluid be leveraged to aid in the diagnosis of patients with subacute or chronic meningitis?\n\nFindings: Metagenomic next-generation sequencing identified parasitic worms, fungi and viruses in a case series of seven subjects. A database of water-only and healthy patient controls enabled application of a z-score based scoring algorithm to effectively separate bona fide pathogen sequences from spurious environmental sequences.\n\nMeaning: Our scoring algorithm greatly simplified data interpretation in a series of patients with a wide range of challenging infectious causes of subacute or chronic meningitis identified by metagenomic next-generation sequencing.

microbiology↗

bioRxiv2017

Silver microwires from treating tooth decay with silver diamine fluoride

Silver diamine fluoride (SDF) is a brush-on treatment for tooth decay that stops 81% of cavitated caries lesions (dental cavities). Before this innovation, caries was treatable only with operative approaches (dental fillings). SDF-treated lesions harden and become resistant to further decay. We hypothesized that the hardening is due to reaction with silver, rather than classic fluoride-mediated remineralization, because infected dentin is not amenable to remineralization. Using synchrotron microCT with 1.3 m resolution, we observe filamentous densities up to 500 m in length and 0.25-7.0 m in diameter, formed in situ by applying SDF to caries lesions. We show that these \"microwires\" fill voids in the lesion caused by disease, and permeate through surrounding dentinal tubules. Using spectroscopy, we confirm that the chemical composition of the observed microwires is predominantly silver. To our knowledge, this represents the first structural microscale observations resulting from clinical SDF treatment. These novel observations hint at mechanistic explanations for the first clinical method to harden carious dentin besides remineralization. We hypothesize that SDF may not only achieve its antimicrobial functions by biochemical interactions, but also through its inherent ability to integrate into dentin.

pharmacology and toxicology↗

bioRxiv2017

Topical Silver Diamine Fluoride For Dental Caries Arrest In Preschool Children: A Randomized Controlled Trial

ObjectivesThe Stopping Cavities Trial investigated effectiveness and safety of 38% silver diamine fluoride in arresting caries lesions.\n\nMaterials and MethodsDouble-blind randomized placebo-controlled superiority trial with 2 parallel groups. Oregon preschools. 66 preschool children with [≥]1 lesion. 38% silver diamine fluoride or placebo (blue-tinted water), applied topically to the lesion. The primary endpoint was caries arrest (lesion inactivity, Nyvad criteria) 14-21 days post intervention. Dental plaque was collected from all children, and microbial composition was assessed by RNA sequencing from 2 lesions and 1 unaffected surface before treatment and at follow-up for 3 children from each group.\n\nResults and ConclusionMean fraction of arrested caries lesions in the silver diamine fluoride group was higher (0.72; 95% CI; 0.55, 0.84) than in the placebo group (0.05; 95% CI; 0.00, 0.16). Confirmatory analysis using generalized estimating equation log-linear regression, accounting for the number of treated surfaces and length of follow-up, indicated the fraction of arrested caries was significantly higher in the treatment group (relative risk, 17.3; 95% CI: 4.3 to 69.4). No harms were observed. RNA sequencing analysis identified no consistent changes in relative abundance of caries-associated microbes, nor emergence of antibiotic or metal resistance gene expression. Topical 38% silver diamine fluoride was effective and safe in arresting cavities in preschool children. The treatment is applicable to primary care practice and may reduce the burden of untreated tooth decay in the population.\n\nTrial RegistrationClinicalTrials.gov NCT02536040.\n\nClinical SignificanceIn this clinical trial, 72% of caries lesions were arrested by silver diamine fluoride, with no harms. Contrary to the presumed antibacterial mechanism, lesion bacterial composition changed negligibly. This simple topical treatment is applicable to primary care practice and may reduce the burden of untreated tooth decay in the population.

clinical trials↗