Julianne R. Brown

Rapid Whole-Genome Sequencing of Mycobacterium tuberculosis Isolates Directly from Clinical Samples.

ABSTRACT The rapid identification of antimicrobial resistance is essential for effective treatment of highly resistant Mycobacterium tuberculosis. Whole-genome sequencing provides comprehensive data on resistance mutations and strain typing for monitoring transmission, but unlike for conventional molecular tests, this has previously been achievable only from cultures of M. tuberculosis. Here we describe a method utilizing biotinylated RNA baits designed specifically for M. tuberculosis DNA to capture full M. tuberculosis genomes directly from infected sputum samples, allowing whole-genome sequencing without the requirement of culture. This was carried out on 24 smear-positive sputum samples, collected from the United Kingdom and Lithuania where a matched culture sample was available, and 2 samples that had failed to grow in culture. M. tuberculosis sequencing data were obtained directly from all 24 smear-positive culture-positive sputa, of which 20 were of high quality (>20× depth and >90% of the genome covered). Results were compared with those of conventional molecular and culture-based methods, and high levels of concordance between phenotypical resistance and predicted resistance based on genotype were observed. High-quality sequence data were obtained from one smear-positive culture-negative case. This study demonstrated for the first time the successful and accurate sequencing of M. tuberculosis genomes directly from uncultured sputa. Identification of known resistance mutations within a week of sample receipt offers the prospect for personalized rather than empirical treatment of drug-resistant tuberculosis, including the use of antimicrobial-sparing regimens, leading to improved outcomes.

Astrovirus VA1/HMO-C: An Increasingly Recognized Neurotropic Pathogen in Immunocompromised Patients

Brain biopsy from a child with unknown cause of encephalopathy was deep-sequenced. Astrovirus VA1/HMO-C was identified, highly divergent from human astroviruses and 95% identical to astrovirus previously associated with encephalitis. Findings suggest astrovirus VA1/HMO-C is an under-recognized cause of viral encephalitis.

Recombination of Globally Circulating Varicella Zoster Virus

ABSTRACT Varicella-zoster virus (VZV) is a human herpesvirus, which during primary infection typically causes varicella (chicken pox) and establishes lifelong latency in sensory and autonomic ganglia. Later in life, the virus may reactivate to cause herpes zoster (HZ; also known as shingles). To prevent these diseases, a live-attenuated heterogeneous vaccine preparation, vOka, is used routinely in many countries worldwide. Recent studies of another alphaherpesvirus, infectious laryngotracheitis virus, demonstrate that live-attenuated vaccine strains can recombine in vivo, creating virulent progeny. These findings raised concerns about using attenuated herpesvirus vaccines under conditions that favor recombination. To investigate whether VZV may undergo recombination, which is a prerequisite for VZV vaccination to create such conditions, we here analyzed 115 complete VZV genomes. Our results demonstrate that recombination occurs frequently for VZV. It thus seems that VZV is fully capable of recombination if given the opportunity, which may have important implications for continued VZV vaccination. Although no interclade vaccine-wild-type recombinant strains were found, intraclade recombinants were frequently detected in clade 2, which harbors the vaccine strains, suggesting that the vaccine strains have already been involved in recombination events, either in vivo or in vitro during passages in cell culture. Finally, previous partial and complete genomic studies have described strains that do not cluster phylogenetically to any of the five established clades. The additional VZV strains sequenced here, in combination with those previously published, have enabled us to formally define a novel sixth VZV clade. IMPORTANCE Although genetic recombination has been demonstrated to frequently occur for other human alphaherpesviruses, herpes simplex viruses 1 and 2, only a few ancient and isolated recent recombination events have hitherto been demonstrated for VZV. In the present study, we demonstrate that VZV also frequently undergoes genetic recombination, including strains belonging to the clade containing the vOKA strain.

Whole-genome enrichment and sequencing of Chlamydia trachomatis directly from clinical samples

BackgroundChlamydia trachomatis is a pathogen of worldwide importance, causing more than 100 million cases of sexually transmitted infections annually. Whole-genome sequencing is a powerful high resolution tool that can be used to generate accurate data on bacterial population structure, phylogeography and mutations associated with antimicrobial resistance. The objective of this study was to perform whole-genome enrichment and sequencing of C. trachomatis directly from clinical samples.MethodsC. trachomatis positive samples comprising seven vaginal swabs and three urine samples were sequenced without prior in vitro culture in addition to nine cultured C. trachomatis samples, representing different serovars. A custom capture RNA bait set, that captures all known diversity amongst C. trachomatis genomes, was used in a whole-genome enrichment step during library preparation to enrich for C. trachomatis DNA. All samples were sequenced on the MiSeq platform.ResultsFull length C. trachomatis genomes (>95-100% coverage of a reference genome) were successfully generated for eight of ten clinical samples and for all cultured samples. The proportion of reads mapping to C. trachomatis and the mean read depth across each genome were strongly linked to the number of bacterial copies within the original sample. Phylogenetic analysis confirmed the known population structure and the data showed potential for identification of minority variants and mutations associated with antimicrobial resistance. The sensitivity of the method was >10-fold higher than other reported methodologies.ConclusionsThe combination of whole-genome enrichment and deep sequencing has proven to be a non-mutagenic approach, capturing all known variation found within C. trachomatis genomes. The method is a consistent and sensitive tool that enables rapid whole-genome sequencing of C. trachomatis directly from clinical samples and has the potential to be adapted to other pathogens with a similar clonal nature.

Human Coronavirus OC43 Associated with Fatal Encephalitis.

In this case report, severe encephalitis with no established cause developed in a child with SCID. Through deep sequencing, human coronavirus OC43 was identified in brain tissue.

Norovirus Whole-Genome Sequencing by SureSelect Target Enrichment: a Robust and Sensitive Method

ABSTRACT Norovirus full-genome sequencing is challenging due to sequence heterogeneity among genomes. Previous methods have relied on PCR amplification, which is problematic due to primer design, and transcriptome sequencing (RNA-Seq), which nonspecifically sequences all RNA, including host and bacterial RNA, in stool specimens. Target enrichment uses a panel of custom-designed 120-mer RNA baits that are complementary to all publicly available norovirus sequences, with multiple baits targeting each position of the genome, which overcomes the challenge of primer design. Norovirus genomes are enriched from stool RNA extracts to minimize the sequencing of nontarget RNA. SureSelect target enrichment and Illumina sequencing were used to sequence full genomes from 507 norovirus-positive stool samples with reverse transcription–real-time PCR cycle threshold (CT ) values of 10 to 43. Sequencing on an Illumina MiSeq system in batches of 48 generated, on average, 81% on-target reads per sample and 100% genome coverage with >12,000-fold read depth. Samples included genotypes GI.1, GI.2, GI.3, GI.6, GI.7, GII.1, GII.2, GII.3, GII.4, GII.5, GII.6, GII.7, GII.13, GII.14, and GII.17. When outliers were accounted for, we generated >80% genome coverage for all positive samples, regardless of CT values. A total of 164 samples were tested in parallel with conventional PCR genotyping of the capsid shell domain; 164/164 samples were successfully sequenced, compared to 158/164 samples that were amplified by PCR. Four of the samples that failed capsid PCR analysis had low titers, which suggests that target enrichment is more sensitive than gel-based PCR. Two samples failed PCR due to primer mismatches; target enrichment uses multiple baits targeting each position, thus accommodating sequence heterogeneity among norovirus genomes.

Norovirus Infections Occur in B-Cell–Deficient Patients

Norovirus incidence was compared between severe combined immunodeficiency children with (n = 10) and without (n = 8) B cells. 60% of B+ and 63% of B- patients developed norovirus infections therefore norovirus replication in B lymphocytes is not essential for infection.

Viral gastrointestinal infections and norovirus genotypes in a paediatric UK hospital, 2014–2015

BACKGROUND Diarrhoea in children is a common disease; understanding the incidence of causative viruses can aid infection control and vaccine development. OBJECTIVES Describe the incidence and characteristics of gastroenteric viruses including norovirus genotypes in a paediatric hospital cohort. STUDY DESIGN Norovirus, adenovirus, sapovirus, astrovirus, rotavirus qPCR and norovirus genotyping results for all stool specimens (n=4786; 1393 patients) at a UK paediatric tertiary referral hospital June 2014-July 2015. RESULTS AND DISCUSSION 24% (329/1393) of patients were positive for a GI virus; the majority were positive for norovirus (44%, 144/329) or adenovirus (44%, 146/329). The overall incidence of rotavirus (2%) is reduced compared to pre-vaccination studies; however the incidence of other GI viruses has not increased. Norovirus infections had a significantly higher virus burden compared to other GI viruses (P ≤0.03); sapovirus infections had the lowest viral burden. The number of norovirus cases per month did not follow the typical winter seasonal trend of nationally reported outbreaks. The number of cases per month correlates with the number of hospital admissions (R=0.703, P=0.011); the number of admissions accounts for 50% of the variability in number of cases per month. The breadth of genotypes seen (48% non-GII.4), suggests a community source for many norovirus infections and has implications for vaccine development. All GI viruses caused chronic infections, with the majority (50-100%) in immunocompromised patients. Incidence or duration of infection in chronic norovirus infections did not differ between genotypes, suggesting host-mediated susceptibility.

The emerging GII.P16-GII.4 Sydney 2012 norovirus lineage is circulating worldwide, arose by late-2014 and contains polymerase changes that may increase virus transmission

Noroviruses are a leading cause of human gastroenteritis worldwide. The norovirus genotype GII.4 is the most prevalent genotype in the human population and has caused six pandemics since 1995. A novel norovirus lineage containing the GII.P16 polymerase and pandemic GII.4 Sydney 2012 capsid was recently detected in Asia and Germany. We demonstrate that this lineage is also circulating within the UK and USA and has been circulating since October 2014 or earlier. While the lineage does not contain unique substitutions in the capsid, it does contain polymerase substitutions close to positions known to influence polymerase function and virus transmission. These polymerase substitutions are shared with a GII.P16-GII.2 virus that dominated outbreaks in Germany in Winter 2016. We suggest that the substitutions in the polymerase may have resulted in a more transmissible virus and the combination of this polymerase and the pandemic GII.4 capsid may result in a highly transmissible virus. Further surveillance efforts will be required to determine whether the GII.P16-GII.4 Sydney 2012 lineage increases in frequency over the coming months.

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.