Rachel Williams

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.

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.

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.

Low Prevalence of Conjunctival Infection with Chlamydia trachomatis in a Treatment-Naïve Trachoma-Endemic Region of the Solomon Islands

Background Trachoma is endemic in several Pacific Island states. Recent surveys across the Solomon Islands indicated that whilst trachomatous inflammation—follicular (TF) was present at levels warranting intervention, the prevalence of trachomatous trichiasis (TT) was low. We set out to determine the relationship between chlamydial infection and trachoma in this population. Methods We conducted a population-based trachoma prevalence survey of 3674 individuals from two Solomon Islands provinces. Participants were examined for clinical signs of trachoma. Conjunctival swabs were collected from all children aged 1–9 years. We tested swabs for Chlamydia trachomatis (Ct) DNA using droplet digital PCR. Chlamydial DNA from positive swabs was enriched and sequenced for use in phylogenetic analysis. Results We observed a moderate prevalence of TF in children aged 1–9 years (n = 296/1135, 26.1%) but low prevalence of trachomatous inflammation—intense (TI) (n = 2/1135, 0.2%) and current Ct infection (n = 13/1002, 1.3%) in children aged 1–9 years, and TT in those aged 15+ years (n = 2/2061, 0.1%). Ten of 13 (76.9%) cases of infection were in persons with TF or TI (p = 0.0005). Sequence analysis of the Ct-positive samples yielded 5/13 (38%) complete (>95% coverage of reference) genome sequences, and 8/13 complete plasmid sequences. Complete sequences all aligned most closely to ocular serovar reference strains. Discussion The low prevalence of TT, TI and Ct infection that we observed are incongruent with the high proportion of children exhibiting signs of TF. TF is present at levels that apparently warrant intervention, but the scarcity of other signs of trachoma indicates the phenotype is mild and may not pose a significant public health threat. Our data suggest that, whilst conjunctival Ct infection appears to be present in the region, it is present at levels that are unlikely to be the dominant driving force for TF in the population. This could be one reason for the low prevalence of TT observed during the study.

Rapid identification of a Mycobacterium tuberculosis full genetic drug resistance profile through whole genome sequencing directly from sputum

INTRODUCTION Resistance to second-line tuberculosis drugs is common, but slow to diagnose with phenotypic drug sensitivity testing. Rapid molecular tests speed up diagnosis, but can only detect limited mutations. Whole genome sequencing (WGS) of culture isolates can generate a complete genetic drug resistance profile, but is delayed by the initial culture step. In the case presented here, successful WGS directly from sputum was achieved using targeted enrichment. CASE REPORT A 29-year-old Nigerian woman was diagnosed with tuberculosis. Xpert MTB/RIF and Hain line probe assays identified rpoB and inhA mutations consistent with rifampicin and intermediate isoniazid resistance, and a further possible mutation conferring fluoroquinolone resistance. WGS directly from sputum identified a further inhA mutation consistent with high-level isoniazid resistance and confirmed the absence of fluoroquinolone resistance. Isoniazid was stopped, and the patient has completed 18 months of a fluoroquinolone-based regimen without relapse. DISCUSSION Compared to rapid molecular tests (which can only examine a limited number of mutations) and WGS of culture isolates (which requires a culture step), WGS directly from sputum can quickly generate a complete genetic drug resistance profile. In this case, WGS altered the clinical management of drug-resistant tuberculosis and demonstrated potential for guiding individualized drug treatment where second-line drug resistance is common.

Detection of Low Frequency Multi-Drug Resistance and Novel Putative Maribavir Resistance in Immunocompromised Pediatric Patients with Cytomegalovirus

Human cytomegalovirus (HCMV) is a significant pathogen in immunocompromised individuals, with the potential to cause fatal pneumonitis and colitis, as well as increasing the risk of organ rejection in transplant patients. With the advent of new anti-HCMV drugs there is therefore considerable interest in using virus sequence data to monitor emerging resistance to antiviral drugs in HCMV viraemia and disease, including the identification of putative new mutations. We used target-enrichment to deep sequence HCMV DNA from 11 immunosuppressed pediatric patients receiving single or combination anti-HCMV treatment, serially sampled over 1–27 weeks. Changes in consensus sequence and resistance mutations were analyzed for three ORFs targeted by anti-HCMV drugs and the frequencies of drug resistance mutations monitored. Targeted-enriched sequencing of clinical material detected mutations occurring at frequencies of 2%. Seven patients showed no evidence of drug resistance mutations. Four patients developed drug resistance mutations a mean of 16 weeks after starting treatment. In two patients, multiple resistance mutations accumulated at frequencies of 20% or less, including putative maribavir and ganciclovir resistance mutations P522Q (UL54) and C480F (UL97). In one patient, resistance was detected 14 days earlier than by PCR. Phylogenetic analysis suggested recombination or superinfection in one patient. Deep sequencing of HCMV enriched from clinical samples excluded resistance in 7 of 11 subjects and identified resistance mutations earlier than conventional PCR-based resistance testing in 2 patients. Detection of multiple low level resistance mutations was associated with poor outcome.

Islands of linkage in an ocean of pervasive recombination reveals two-speed evolution of human cytomegalovirus genomes

Abstract Human cytomegalovirus (HCMV) infects most of the population worldwide, persisting throughout the hosts life in a latent state with periodic episodes of reactivation. While typically asymptomatic, HCMV can cause fatal disease among congenitally infected infants and immunocompromised patients. These clinical issues are compounded by the emergence of antiviral resistance and the absence of an effective vaccine, the development of which is likely complicated by the numerous immune evasins encoded by HCMV to counter the hosts adaptive immune responses, a feature that facilitates frequent super-infections. Understanding the evolutionary dynamics of HCMV is essential for the development of effective new drugs and vaccines. By comparing viral genomes from uncultivated or low-passaged clinical samples of diverse origins, we observe evidence of frequent homologous recombination events, both recent and ancient, and no structure of HCMV genetic diversity at the whole-genome scale. Analysis of individual gene-scale loci reveals a striking dichotomy: while most of the genome is highly conserved, recombines essentially freely and has evolved under purifying selection, 21 genes display extreme diversity, structured into distinct genotypes that do not recombine with each other. Most of these hyper-variable genes encode glycoproteins involved in cell entry or escape of host immunity. Evidence that half of them have diverged through episodes of intense positive selection suggests that rapid evolution of hyper-variable loci is likely driven by interactions with host immunity. It appears that this process is enabled by recombination unlinking hyper-variable loci from strongly constrained neighboring sites. It is conceivable that viral mechanisms facilitating super-infection have evolved to promote recombination between diverged genotypes, allowing the virus to continuously diversify at key loci to escape immune detection, while maintaining a genome optimally adapted to its asymptomatic infectious lifecycle.

On the effective depth of viral sequence data.

Abstract Genome sequence data are of great value in describing evolutionary processes in viral populations. However, in such studies, the extent to which data accurately describes the viral population is a matter of importance. Multiple factors may influence the accuracy of a dataset, including the quantity and nature of the sample collected, and the subsequent steps in viral processing. To investigate this phenomenon, we sequenced replica datasets spanning a range of viruses, and in which the point at which samples were split was different in each case, from a dataset in which independent samples were collected from a single patient to another in which all processing steps up to sequencing were applied to a single sample before splitting the sample and sequencing each replicate. We conclude that neither a high read depth nor a high template number in a sample guarantee the precision of a dataset. Measures of consistency calculated from within a single biological sample may also be insufficient; distortion of the composition of a population by the experimental procedure or genuine within-host diversity between samples may each affect the results. Where it is possible, data from replicate samples should be collected to validate the consistency of short-read sequence data.

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.

Direct whole genome sequencing of sputum accurately identifies drug resistant Mycobacterium tuberculosis faster than MGIT culture sequencing

The current methods available to diagnose antimicrobial-resistant Mycobacterium tuberculosis infections require a positive culture or only test a limited number of resistance-associated mutations. A rapid accurate identification of antimicrobial resistance enables the prompt initiation of effective treatment. ABSTRACT The current methods available to diagnose antimicrobial-resistant Mycobacterium tuberculosis infections require a positive culture or only test a limited number of resistance-associated mutations. A rapid accurate identification of antimicrobial resistance enables the prompt initiation of effective treatment. Here, we determine the utility of whole-genome sequencing (WGS) of M. tuberculosis directly from routinely obtained diagnostic sputum samples to provide a comprehensive resistance profile compared to that from mycobacterial growth indicator tube (MGIT) WGS. We sequenced M. tuberculosis from 43 sputum samples by targeted DNA enrichment using the Agilent SureSelectXT kit, and 43 MGIT positive samples from each participant. Thirty two (74%) sputum samples and 43 (100%) MGIT samples generated whole genomes. The times to antimicrobial resistance profiles and concordance were compared with Xpert MTB/RIF and phenotypic resistance testing from cultures of the same samples. Antibiotic susceptibility could be predicted from WGS of sputum within 5 days of sample receipt and up to 24 days earlier than WGS from MGIT culture and up to 31 days earlier than phenotypic testing. Direct sputum results could be reduced to 3 days with faster hybridization and if only regions encoding drug resistance are sequenced. We show that direct sputum sequencing has the potential to provide comprehensive resistance detection significantly faster than MGIT whole-genome sequencing or phenotypic testing of resistance from cultures in a clinical setting. This improved turnaround time enables prompt appropriate treatment with associated patient and health service benefits. Improvements in sample preparation are necessary to ensure comparable sensitivities and complete resistance profile predictions in all cases.