Untargeted metagenomics protocol for the diagnosis of infection from CSF and tissue from sterile sites.

Metagenomic next-generation sequencing (mNGS) is an untargeted technique capable of detecting all microbial nucleic acid within a sample. This protocol outlines our wet laboratory method for mNGS of cerebrospinal fluid (CSF) specimens and tissues from sterile sites. We use this method routinely in our clinical service, processing 178 specimens over the past 2.5 years in a laboratory that adheres to ISO:15189 standards. We have successfully used this protocol to diagnose multiple cases of encephalitis and hepatitis.

Genomic investigations of unexplained acute hepatitis in children.

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children.

Epstein-Barr virus (EBV) deletions as biomarkers of response to treatment of chronic active EBV.

Chronic active Epstein-Barr virus (CAEBV) disease is a rare condition characterised by persistent EBV infection in previously healthy individuals. Defective EBV genomes were found in East Asian patients with CAEBV. In the present study, we sequenced 14 blood EBV samples from three UK patients with CAEBV, comparing the results with saliva CAEBV samples and other conditions. We observed EBV deletions in blood, some of which may disrupt viral replication, but not saliva in CAEBV. Deletions were lost overtime after successful treatment. These findings are compatible with CAEBV being associated with the evolution and persistence of EBV+ haematological clones that are lost on successful treatment.

Benchmark of thirteen bioinformatic pipelines for metagenomic virus diagnostics using datasets from clinical samples.

INTRODUCTION: Metagenomic sequencing is increasingly being used in clinical settings for difficult to diagnose cases. The performance of viral metagenomic protocols relies to a large extent on the bioinformatic analysis. In this study, the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS) initiated a benchmark of metagenomic pipelines currently used in clinical virological laboratories. METHODS: Metagenomic datasets from 13 clinical samples from patients with encephalitis or viral respiratory infections characterized by PCR were selected. The datasets were analyzed with 13 different pipelines currently used in virological diagnostic laboratories of participating ENNGS members. The pipelines and classification tools were: Centrifuge, DAMIAN, DIAMOND, DNASTAR, FEVIR, Genome Detective, Jovian, MetaMIC, MetaMix, One Codex, RIEMS, VirMet, and Taxonomer. Performance, characteristics, clinical use, and user-friendliness of these pipelines were analyzed. RESULTS: Overall, viral pathogens with high loads were detected by all the evaluated metagenomic pipelines. In contrast, lower abundance pathogens and mixed infections were only detected by 3/13 pipelines, namely DNASTAR, FEVIR, and MetaMix. Overall sensitivity ranged from 80% (10/13) to 100% (13/13 datasets). Overall positive predictive value ranged from 71-100%. The majority of the pipelines classified sequences based on nucleotide similarity (8/13), only a minority used amino acid similarity, and 6 of the 13 pipelines assembled sequences de novo. No clear differences in performance were detected that correlated with these classification approaches. Read counts of target viruses varied between the pipelines over a range of 2-3 log, indicating differences in limit of detection. CONCLUSION: A wide variety of viral metagenomic pipelines is currently used in the participating clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implicating the need for standardization and validation of metagenomic analysis for clinical diagnostic use. Future studies should address the selective effects due to the choice of different reference viral databases.

The influence of human genetic variation on Epstein-Barr virus sequence diversity.

Epstein-Barr virus (EBV) is one of the most common viruses latently infecting humans. Little is known about the impact of human genetic variation on the large inter-individual differences observed in response to EBV infection. To search for a potential imprint of host genomic variation on the EBV sequence, we jointly analyzed paired viral and human genomic data from 268 HIV-coinfected individuals with CD4 + T cell count < 200/mm3 and elevated EBV viremia. We hypothesized that the reactivated virus circulating in these patients could carry sequence variants acquired during primary EBV infection, thereby providing a snapshot of early adaptation to the pressure exerted on EBV by the individual immune response. We searched for associations between host and pathogen genetic variants, taking into account human and EBV population structure. Our analyses revealed significant associations between human and EBV sequence variation. Three polymorphic regions in the human genome were found to be associated with EBV variation: one at the amino acid level (BRLF1:p.Lys316Glu); and two at the gene level (burden testing of rare variants in BALF5 and BBRF1). Our findings confirm that jointly analyzing host and pathogen genomes can identify sites of genomic interactions, which could help dissect pathogenic mechanisms and suggest new therapeutic avenues.

Mixed cytomegalovirus genotypes in HIV-positive mothers show compartmentalization and distinct patterns of transmission to infants.

Cytomegalovirus (CMV) is the commonest cause of congenital infection and particularly so among infants born to HIV-infected women. Studies of congenital CMV infection (cCMVi) pathogenesis are complicated by the presence of multiple infecting maternal CMV strains, especially in HIV-positive women, and the large, recombinant CMV genome. Using newly developed tools to reconstruct CMV haplotypes, we demonstrate anatomic CMV compartmentalization in five HIV-infected mothers and identify the possibility of congenitally transmitted genotypes in three of their infants. A single CMV strain was transmitted in each congenitally infected case, and all were closely related to those that predominate in the cognate maternal cervix. Compared to non-transmitted strains, these congenitally transmitted CMV strains showed statistically significant similarities in 19 genes associated with tissue tropism and immunomodulation. In all infants, incident superinfections with distinct strains from breast milk were captured during follow-up. The results represent potentially important new insights into the virologic determinants of early CMV infection.

STROBE-metagenomics: a STROBE extension statement to guide the reporting of metagenomics studies.

The term metagenomics refers to the use of sequencing methods to simultaneously identify genomic material from all organisms present in a sample, with the advantage of greater taxonomic resolution than culture or other methods. Applications include pathogen detection and discovery, species characterisation, antimicrobial resistance detection, virulence profiling, and study of the microbiome and microecological factors affecting health. However, metagenomics involves complex and multistep processes and there are important technical and methodological challenges that require careful consideration to support valid inference. We co-ordinated a multidisciplinary, international expert group to establish reporting guidelines that address specimen processing, nucleic acid extraction, sequencing platforms, bioinformatics considerations, quality assurance, limits of detection, power and sample size, confirmatory testing, causality criteria, cost, and ethical issues. The guidance recognises that metagenomics research requires pragmatism and caution in interpretation, and that this field is rapidly evolving.

Metagenomic analysis of a blood stain from the French revolutionary Jean-Paul Marat (1743-1793).

The French revolutionary Jean-Paul Marat (1743-1793) was assassinated in 1793 in his bathtub, where he was trying to find relief from the debilitating skin disease he was suffering from. At the time of his death, Marat was annotating newspapers, which got stained with his blood and were subsequently preserved by his sister. We extracted and sequenced DNA from the blood stain and also from another section of the newspaper, which we used for comparison. Results from the human DNA sequence analyses were compatible with a heterogeneous ancestry of Marat, with his mother being of French origin and his father born in Sardinia. Metagenomic analyses of the non-human reads uncovered the presence of fungal, bacterial and low levels of viral DNA. Relying on the presence/absence of microbial species in the samples, we could cast doubt on several putative infectious agents that have been previously hypothesised as the cause of his condition but for which we detect not a single sequencing read. Conversely, some of the species we detect are uncommon as environmental contaminants and may represent plausible infective agents. Based on all the available evidence, we hypothesize that Marat may have suffered from a fungal infection (seborrheic dermatitis), possibly superinfected with bacterial opportunistic pathogens.

Use of Whole-Genome Sequencing of Adenovirus in Immunocompromised Pediatric Patients to Identify Nosocomial Transmission and Mixed-Genotype Infection.

Background: Adenoviruses are significant pathogens for the immunocompromised, arising from primary infection or reinfection. Serotyping is insufficient to support nosocomial transmission investigations. We investigate whether whole-genome sequencing (WGS) provides clinically relevant information on transmission among patients in a pediatric tertiary hospital. Methods: We developed a target-enriched adenovirus WGS technique for clinical samples and retrospectively sequenced 107 adenovirus-positive residual diagnostic samples, including viremias (>5 × 104 copies/mL), from 37 patients collected January 2011-March 2016. Whole-genome sequencing was used to determine genotype and for phylogenetic analysis. Results: Adenovirus sequences were recovered from 105 of 107 samples. Full genome sequences were recovered from all 20 nonspecies C samples and from 36 of 85 species C viruses, with partial genome sequences recovered from the rest. Whole-genome phylogenetic analysis suggested linkage of 3 genotype A31 cases and uncovered an unsuspected epidemiological link to an A31 infection first detected on the same ward 4 years earlier. In 9 samples from 1 patient who died, we identified a mixed genotype adenovirus infection. Conclusions: Adenovirus WGS from clinical samples is possible and useful for genotyping and molecular epidemiology. Whole-genome sequencing identified likely nosocomial transmission with greater resolution than conventional genotyping and distinguished between adenovirus disease due to single or multiple genotypes.

Deep sequencing reveals persistence of cell-associated mumps vaccine virus in chronic encephalitis.

Routine childhood vaccination against measles, mumps and rubella has virtually abolished virus-related morbidity and mortality. Notwithstanding this, we describe here devastating neurological complications associated with the detection of live-attenuated mumps virus Jeryl Lynn (MuVJL5) in the brain of a child who had undergone successful allogeneic transplantation for severe combined immunodeficiency (SCID). This is the first confirmed report of MuVJL5 associated with chronic encephalitis and highlights the need to exclude immunodeficient individuals from immunisation with live-attenuated vaccines. The diagnosis was only possible by deep sequencing of the brain biopsy. Sequence comparison of the vaccine batch to the MuVJL5 isolated from brain identified biased hypermutation, particularly in the matrix gene, similar to those found in measles from cases of SSPE. The findings provide unique insights into the pathogenesis of paramyxovirus brain infections.

An emerging opportunistic infection: fatal astrovirus (VA1/HMO-C) encephalitis in a pediatric stem cell transplant recipient.

Neuroinvasive astrovirus (VA1-HMO-C) is an emerging life-threatening infection in immunocompromised hosts. We describe an 8-month-old child who died of VA1/HMO-C encephalitis following bone marrow transplantation. The diagnosis was only made post-mortem using RNA deep sequencing of the brain. Repeat analysis of the post-mortem brain tissue using polymerase chain reaction specific primers for VA1/HMO-C was positive. Astrovirus VA1/HMO-C should be included in the evaluation of patients with similar encephalitis.

Human IFNAR2 deficiency: Lessons for antiviral immunity.

Type I interferon (IFN-α/ß) is a fundamental antiviral defense mechanism. Mouse models have been pivotal to understanding the role of IFN-α/ß in immunity, although validation of these findings in humans has been limited. We investigated a previously healthy child with fatal encephalitis after inoculation of the live attenuated measles, mumps, and rubella (MMR) vaccine. By targeted resequencing, we identified a homozygous mutation in the high-affinity IFN-α/ß receptor (IFNAR2) in the proband, as well as a newborn sibling, that rendered cells unresponsive to IFN-α/ß. Reconstitution of the proband's cells with wild-type IFNAR2 restored IFN-α/ß responsiveness and control of IFN-attenuated viruses. Despite the severe outcome of systemic live vaccine challenge, the proband had previously shown no evidence of heightened susceptibility to respiratory viral pathogens. The phenotype of IFNAR2 deficiency, together with similar findings in STAT2-deficient patients, supports an essential but narrow role for IFN-α/ß in human antiviral immunity.

Novel Human Embryonic Stem Cell Regulators Identified by Conserved and Distinct CpG Island Methylation State.

Human embryonic stem cells (hESCs) undergo epigenetic changes in vitro which may compromise function, so an epigenetic pluripotency "signature" would be invaluable for line validation. We assessed Cytosine-phosphate-Guanine Island (CGI) methylation in hESCs by genomic DNA hybridisation to a CGI array, and saw substantial variation in CGI methylation between lines. Comparison of hESC CGI methylation profiles to corresponding somatic tissue data and hESC mRNA expression profiles identified a conserved hESC-specific methylation pattern associated with expressed genes. Transcriptional repressors and activators were over-represented amongst genes whose associated CGIs were methylated or unmethylated specifically in hESCs, respectively. Knockdown of candidate transcriptional regulators (HMGA1, GLIS2, PFDN5) induced differentiation in hESCs, whereas ectopic expression in fibroblasts modulated iPSC colony formation. Chromatin immunoprecipitation confirmed interaction between the candidates and the core pluripotency transcription factor network. We thus identify novel pluripotency genes on the basis of a conserved and distinct epigenetic configuration in human stem cells.

Bayesian mixture analysis for metagenomic community profiling.

MOTIVATION: Deep sequencing of clinical samples is now an established tool for the detection of infectious pathogens, with direct medical applications. The large amount of data generated produces an opportunity to detect species even at very low levels, provided that computational tools can effectively profile the relevant metagenomic communities. Data interpretation is complicated by the fact that short sequencing reads can match multiple organisms and by the lack of completeness of existing databases, in particular for viral pathogens. Here we present metaMix, a Bayesian mixture model framework for resolving complex metagenomic mixtures. We show that the use of parallel Monte Carlo Markov chains for the exploration of the species space enables the identification of the set of species most likely to contribute to the mixture. RESULTS: We demonstrate the greater accuracy of metaMix compared with relevant methods, particularly for profiling complex communities consisting of several related species. We designed metaMix specifically for the analysis of deep transcriptome sequencing datasets, with a focus on viral pathogen detection; however, the principles are generally applicable to all types of metagenomic mixtures. AVAILABILITY AND IMPLEMENTATION: metaMix is implemented as a user friendly R package, freely available on CRAN: http://cran.r-project.org/web/packages/metaMix CONTACT: sofia.morfopoulou.10@ucl.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bionformatics online.

Astrovirus VA1/HMO-C: an increasingly recognized neurotropic pathogen in immunocompromised patients.

BACKGROUND: An 18-month-old boy developed encephalopathy, for which extensive investigation failed to identify an etiology, 6 weeks after stem cell transplant. To exclude a potential infectious cause, we performed high-throughput RNA sequencing on brain biopsy. METHODS: RNA-Seq was performed on an Illumina Miseq, generating 20 million paired-end reads. Nonhost data were checked for similarity to known organisms using BLASTx. The full viral genome was sequenced by primer walking. RESULTS: We identified an astrovirus, HAstV-VA1/HMO-C-UK1(a), which was highly divergent from human astrovirus (HAstV 1-8) genotypes, but closely related to VA1/HMO-C astroviruses, including one recovered from a case of fatal encephalitis in an immunosuppressed child. The virus was detected in stool and serum, with highest levels in brain and cerebrospinal fluid (CSF). Immunohistochemistry of the brain biopsy showed positive neuronal staining. A survey of 680 stool and 349 CSF samples identified a related virus in the stool of another immunosuppressed child. CONCLUSIONS: The discovery of HAstV-VA1/HMO-C-UK1(a) as the cause of encephalitis in this case provides further evidence that VA1/HMO-C viruses, unlike HAstV 1-8, are neuropathic, particularly in immunocompromised patients, and should be considered in the differential diagnosis of encephalopathy. With a turnaround from sample receipt to result of <1 week, we confirm that RNA-Seq presents a valuable diagnostic tool in unexplained encephalitis.

Oct4 and LIF/Stat3 additively induce Krüppel factors to sustain embryonic stem cell self-renewal.

Embryonic stem cell (ESC) pluripotency is dependent on an intrinsic gene regulatory network centered on Oct4. Propagation of the pluripotent state is stimulated by the cytokine leukemia inhibitory factor (LIF) acting through the transcriptional regulator Stat3. Here, we show that this extrinsic stimulus converges with the intrinsic circuitry in Krüppel-factor activation. Oct4 primarily induces Klf2 while LIF/Stat3 selectively enhances Klf4 expression. Overexpression of either factor reduces LIF dependence, but with quantitative and qualitative differences. Unlike Klf4, Klf2 increases ESC clonogenicity, maintains undifferentiated ESCs in the genetic absence of Stat3, and confers resistance to BMP-induced differentiation. ESCs expanded with Klf2 remain capable of contributing to adult chimeras. Postimplantation-embryo-derived EpiSCs lack both Klf2 and Klf4 and expression of either can reinstate naive pluripotency. These findings indicate that Oct4 and Stat3 intersect in directing expression of Klf transcriptional regulators with overlapping properties that additively reinforce ground-state ESC pluripotency, identity, and self-renewal.