Identification of two novel subtypes of hepatitis C virus genotype 8 and a potential new genotype successfully treated with direct acting antivirals.

BACKGROUND: Hepatitis C virus (HCV) has a high genetic diversity and is classified into 8 genotypes and over 90 subtypes with some endemic to specific world regions. This could compromise direct-acting antiviral (DAA) efficacy and global HCV elimination. METHODS: We characterised HCV subtypes 'rare' to the UK (non-1a/1b/2b/3a/4d) by whole genome sequencing via a national surveillance programme. Genetic analyses to determine the genotype of samples with unresolved genotypes were undertaken by comparison with ICTV HCV reference sequences. RESULTS: Two HCV variants were characterised as being closely related to the recently identified genotype 8 (GT8), with >85% pairwise genetic distance similarity to GT8 sequences and within the typical inter-subtype genetic distance range. The individuals infected by the variants were UK residents originally from Pakistan and India. In contrast, a third variant was only confidently identified to be more similar to GT6 compared to other genotypes across 6% of the genome and was isolated from a UK resident originally from Guyana. All three were cured with pangenotypic DAAs (Sofosbuvir + Velpatasvir or Glecaprevir + Pibrentasvir) despite the presence of resistance polymorphisms in NS3 (80 K/168E), NS5A (28 V/30S/62L/92S/93S) and NS5B (159F). CONCLUSIONS: This study expands our knowledge of HCV diversity by identifying two new GT8 subtypes and potentially a new genotype.

Clinical evaluation of a Hepatitis C Virus whole-genome sequencing pipeline for genotyping and resistance testing.

OBJECTIVES: We sought to evaluate clinically a hepatitis C virus (HCV) whole-genome, next-generation sequencing (NGS) pipeline that is agnostic to viral genotype. METHODS: Performance of the NGS pipeline was assessed through comparison of results with Sanger sequencing (SS) of partial HCV genomes. RESULTS: There was 98.7% (376/381) concordance for viral subtype between SS and NGS. The positive and negative per cent agreements for determination of resistance-associated substitutions were 97.8% (95% CI 92.5-99.4%) and 99.9% (95% CI 99.5-100.0%), respectively. The NGS pipeline was also able to detect novel subtypes, mixtures, recombinants, transiently occurring resistance mutations and distinguish re-infection with the same subtype from relapse. DISCUSSION: Particular scenarios where NGS may be used include settings without universal access to pan-genotypic antiviral regimens, those infected with a 'rare' subtype or who have been failed by first-line therapy, and in cases of suspected re-infection.

Coevolved Multidrug-Resistant HIV-1 Protease and Reverse Transcriptase Influences Integrase Drug Susceptibility and Replication Fitness.

Integrase strand transfer inhibitors (InSTIs) are recommended agents in first-line combination antiretroviral therapy (cART). We examined the evolution of drug resistance mutations throughout HIV-1 pol and the effects on InSTI susceptibility and viral fitness. We performed single-genome sequencing of full-length HIV-1 pol in a highly treatment-experienced patient, and determined drug susceptibility of patient-derived HIV-1 genomes using a phenotypic assay encompassing full-length pol gene. We show the genetic linkage of multiple InSTI-resistant haplotypes containing major resistance mutations at Y143, Q148 and N155 to protease inhibitor (PI) and reverse transcriptase inhibitor (RTI) resistance mutations. Phenotypic analysis of viruses expressing patient-derived IN genes with eight different InSTI-resistant haplotypes alone or in combination with coevolved protease (PR) and RT genes exhibited similar levels of InSTI susceptibility, except for three haplotypes that showed up to 3-fold increases in InSTI susceptibility (p ≤ 0.032). The replicative fitness of most viruses expressing patient-derived IN only significantly decreased, ranging from 8% to 56% (p ≤ 0.01). Interestingly, the addition of coevolved PR + RT significantly increased the replicative fitness of some haplotypes by up to 73% (p ≤ 0.024). Coevolved PR + RT contributes to the susceptibility and viral fitness of patient-derived IN viruses. Maintaining patients on failing cART promotes the selection of fitter resistant strains, and thereby limits future therapy options.

Real world SOF/VEL/VOX retreatment outcomes and viral resistance analysis for HCV patients with prior failure to DAA therapy.

Sustained viral response (SVR) rates for direct-acting antiviral (DAA) therapy for hepatitis C virus (HCV) infection routinely exceed 95%. However, a small number of patients require retreatment. Sofosbuvir, velpatasvir and voxilaprevir (SOF/VEL/VOX) is a potent DAA combination primarily used for the retreatment of patients who failed by DAA therapies. Here we evaluate retreatment outcomes and the effects of resistance-associated substitutions (RAS) in a real-world cohort, including a large number of genotype (GT)3 infected patients. 144 patients from the UK were retreated with SOF/VEL/VOX following virologic failure with first-line DAA treatment regimens. Full-length HCV genome sequencing was performed prior to retreatment with SOF/VEL/VOX. HCV subtypes were assigned and RAS relevant to each genotype were identified. GT1a and GT3a each made up 38% (GT1a n = 55, GT3a n = 54) of the cohort. 40% (n = 58) of patients had liver cirrhosis of whom 7% (n = 4) were decompensated, 10% (n = 14) had hepatocellular carcinoma (HCC) and 8% (n = 12) had received a liver transplant prior to retreatment. The overall retreatment SVR12 rate was 90% (129/144). On univariate analysis, GT3 infection (50/62; SVR = 81%, p = .009), cirrhosis (47/58; SVR = 81%, p = .01) and prior treatment with SOF/VEL (12/17; SVR = 71%, p = .02) or SOF+DCV (14/19; SVR = 74%, p = .012) were significantly associated with retreatment failure, but existence of pre-retreatment RAS was not when viral genotype was taken into account. Retreatment with SOF/VEL/VOX is very successful for non-GT3-infected patients. However, for GT3-infected patients, particularly those with cirrhosis and failed by initial SOF/VEL treatment, SVR rates were significantly lower and alternative retreatment regimens should be considered.

Technical Validation of a Hepatitis C Virus Whole Genome Sequencing Assay for Detection of Genotype and Antiviral Resistance in the Clinical Pathway.

Choice of direct acting antiviral (DAA) therapy for Hepatitis C Virus (HCV) in the United Kingdom and similar settings usually requires knowledge of the genotype and, in some cases, antiviral resistance (AVR) profile of the infecting virus. To determine these, most laboratories currently use Sanger technology, but next-generation sequencing (NGS) offers potential advantages in throughput and accuracy. However, NGS poses unique technical challenges, which require idiosyncratic development and technical validation approaches. This applies particularly to virology, where sequence diversity is high and the amount of starting genetic material is low, making it difficult to distinguish real data from artifacts. We describe the development and technical validation of a sequence capture-based HCV whole genome sequencing (WGS) assay to determine viral genotype and AVR profile. We use clinical samples of known subtypes and viral loads, and simulated FASTQ datasets to validate the analytical performances of both the wet laboratory and bioinformatic pipeline procedures. We show high concordance of the WGS assay compared to current "gold standard" Sanger assays. Specificity was 92.3 and 96.1% for AVR and genotyping, respectively. Discordances were due to the inability of Sanger assays to assign the correct subtype or accurately call mixed drug-resistant variants. We show high repeatability and reproducibility with >99.8% sequence similarity between sequence runs as well as high precision for variant frequency detection at >98.8% in the 95th percentile. Post-sequencing bioinformatics quality control workflows allow the accurate distinction between mixed infections, cross-contaminants and recombinant viruses at a threshold of >5% for the minority population. The sequence capture-based HCV WGS assay is more accurate than legacy AVR and genotyping assays. The assay has now been implemented in the clinical pathway of England's National Health Service HCV treatment programs, representing the first validated HCV WGS pipeline in clinical service. The data generated will additionally provide granular national-level genomic information for public health policy making and support the WHO HCV elimination strategy.

Enhanced Detection of DNA Viruses in the Cerebrospinal Fluid of Encephalitis Patients Using Metagenomic Next-Generation Sequencing.

The long and expanding list of viral pathogens associated with causing encephalitis confounds current diagnostic procedures, and in up to 50% of cases, the etiology remains undetermined. Sequence-agnostic metagenomic next-generation sequencing (mNGS) obviates the need to specify targets in advance and thus has great potential in encephalitis diagnostics. However, the low relative abundance of viral nucleic acids in clinical specimens poses a significant challenge. Our protocol employs two novel techniques to selectively remove human material at two stages, significantly increasing the representation of viral material. Our bioinformatic workflow using open source protein- and nucleotide sequence-matching software balances sensitivity and specificity in diagnosing and characterizing any DNA viruses present. A panel of 12 cerebrospinal fluid (CSFs) from encephalitis cases was retrospectively interrogated by mNGS, with concordant results in seven of nine samples with a definitive DNA virus diagnosis, and a different herpesvirus was identified in the other two. In two samples with an inconclusive diagnosis, DNA viruses were detected and in a virus-negative sample, no viruses were detected. This assay has the potential to detect DNA virus infections in cases of encephalitis of unknown etiology and to improve the current screening tests by identifying new and emerging agents.

Surveillance of HIV-1 transmitted integrase strand transfer inhibitor resistance in the UK.

BACKGROUND: HIV treatment guidelines have traditionally recommended that all HIV-positive individuals are tested for evidence of drug resistance prior to starting ART. Testing for resistance to reverse transcriptase inhibitors and PIs is well established in routine care. However, testing for integrase strand transfer inhibitor (InSTI) resistance is less consistent. OBJECTIVES: To inform treatment guidelines by determining the prevalence of InSTI resistance in a national cohort of recently infected individuals. PATIENTS AND METHODS: Recent (within 4 months) HIV-1 infections were identified using a Recent Infection Testing Algorithm of new HIV-1 diagnoses in the UK. Resistance-associated mutations (RAMs) in integrase, protease and reverse transcriptase were detected by ultradeep sequencing, which allows for the sensitive estimation of the frequency of each resistant variant in a sample. RESULTS: The analysis included 655 randomly selected individuals (median age = 33 years, 95% male, 83% MSM, 78% white) sampled in the period 2014 to 2016 and determined to have a recent infection. These comprised 320, 138 and 197 samples from 2014, 2015 and 2016, respectively. None of the samples had major InSTI RAMs occurring at high variant frequency (≥20%). A subset (25/640, 3.9%) had major InSTI RAMs occurring only as low-frequency variants (2%-20%). In contrast, 47/588 (8.0%) had major reverse transcriptase inhibitor and PI RAMs at high frequency. CONCLUSIONS: Between 2014 and 2016, major InSTI RAMs were uncommon in adults with recent HIV-1 infection, only occurring as low-frequency variants of doubtful clinical significance. Continued surveillance of newly diagnosed patients for evidence of transmitted InSTI resistance is recommended to inform clinical practice.

Multi-Laboratory Comparison of Next-Generation to Sanger-Based Sequencing for HIV-1 Drug Resistance Genotyping.

Next-generation sequencing (NGS) is increasingly used for HIV-1 drug resistance genotyping. NGS methods have the potential for a more sensitive detection of low-abundance variants (LAV) compared to standard Sanger sequencing (SS) methods. A standardized threshold for reporting LAV that generates data comparable to those derived from SS is needed to allow for the comparability of data from laboratories using NGS and SS. Ten HIV-1 specimens were tested in ten laboratories using Illumina MiSeq-based methods. The consensus sequences for each specimen using LAV thresholds of 5%, 10%, 15%, and 20% were compared to each other and to the consensus of the SS sequences (protease 4-99; reverse transcriptase 38-247). The concordance among laboratories' sequences at different thresholds was evaluated by pairwise sequence comparisons. NGS sequences generated using the 20% threshold were the most similar to the SS consensus (average 99.6% identity, range 96.1-100%), compared to 15% (99.4%, 88.5-100%), 10% (99.2%, 87.4-100%), or 5% (98.5%, 86.4-100%). The average sequence identity between laboratories using thresholds of 20%, 15%, 10%, and 5% was 99.1%, 98.7%, 98.3%, and 97.3%, respectively. Using the 20% threshold, we observed an excellent agreement between NGS and SS, but significant differences at lower thresholds. Understanding how variation in NGS methods influences sequence quality is essential for NGS-based HIV-1 drug resistance genotyping.

Challenges of aciclovir-resistant HSV infection in allogeneic bone marrow transplant recipients.

INTRODUCTION: The emergence of herpes simplex virus (HSV) resistance to aciclovir (ACV) has increasingly been reported among hematopoietic stem cell transplant (HSCT) recipients and often associated with extended ACV prophylaxis. METHODS: Between June 2011 and June 2019, medical records of 532 HSCT recipients with suspected HSV infection were retrospectively analyzed. HSV-1 and HSV-2 positive samples were identified in 47 and 16 patients respectively. Analysis of HSV resistance to antivirals was performed at the Public Health England reference laboratory in London using phenotypic and/or genotypic resistance assays. RESULTS: The prevalence of ACV-resistant HSV accounted for 17% (8/48) of infected HSV-1 cases. All 8 patients received T-cell depleted allogeneic HSCT for hematological malignancies. Half of these patients were male with a median age was 57.5 years (range; 26-63). Chronic Graft versus Host disease (cGVHD) affected 7 patients before HSV-1 diagnosis. HSV-1 infection developed while receiving either intravenous ACV (n = 2) or oral ACV (n = 6 patients) prophylaxis at a median of 373 [range,18-2183] days post-HSCT. ACV resistance was clinically suspected at a median of 25 [range,16-109] days after initial HSV diagnosis and subsequently laboratory confirmed at a median of 25 (range,10-59) days. All patients presented with hemorrhagic oral mucositis refractory to treatment dose ACV. Foscarnet (FOS) treatment was initiated in all 8 patients (pending laboratory confirmation of ACV resistance) with some effect but associated with significant toxicity burden. Four patients presented again with recurrent HSV infection or no resolution. Three with recurrent HSV died from other causes while suffering from persistent oral HSV lesions. CONCLUSION: A prolonged immunosuppressed state following T-deplete HSCTs alongside extended use of ACV, early onset systemic HSV infection, presence of cGVHD, and treatment toxicities pose a significant challenge to the management of ACV resistant HSV infections and alternative effective antiviral options remains an unmet need in this clinical setting.

Consensus recommendations for resistance testing in the management of chronic hepatitis C virus infection: Public Health England HCV Resistance Group.

The treatment of hepatitis C virus (HCV) infection has been revolutionised by the advent of oral, well-tolerated, direct acting antiviral therapies (DAA), with high cure rates. However, in some scenarios, HCV resistance to antiviral therapies may have an impact on treatment success. Public Health England's HCV Resistance Group was established to support clinicians treating people with HCV, where the issue of resistance may be a factor in clinical decision-making, and this review includes the Group's current recommendations on the use of HCV resistance testing. The authors describe the principles behind and approach to HCV resistance testing and consider evidence from in vitro studies, clinical trials and real world cohorts on the impact of HCV resistance on treatment outcomes for particular DAA regimens. Five scenarios are identified in the UK and similar settings, where, in the Group's opinion, resistance testing should be performed.

Interpreting Viral Deep Sequencing Data with GLUE.

Using deep sequencing technologies such as Illumina's platform, it is possible to obtain reads from the viral RNA population revealing the viral genome diversity within a single host. A range of software tools and pipelines can transform raw deep sequencing reads into Sequence Alignment Mapping (SAM) files. We propose that interpretation tools should process these SAM files, directly translating individual reads to amino acids in order to extract statistics of interest such as the proportion of different amino acid residues at specific sites. This preserves per-read linkage between nucleotide variants at different positions within a codon location. The samReporter is a subsystem of the GLUE software toolkit which follows this direct read translation approach in its processing of SAM files. We test samReporter on a deep sequencing dataset obtained from a cohort of 241 UK HCV patients for whom prior treatment with direct-acting antivirals has failed; deep sequencing and resistance testing have been suggested to be of clinical use in this context. We compared the polymorphism interpretation results of the samReporter against an approach that does not preserve per-read linkage. We found that the samReporter was able to properly interpret the sequence data at resistance-associated locations in nine patients where the alternative approach was equivocal. In three cases, the samReporter confirmed that resistance or an atypical substitution was present at NS5A position 30. In three further cases, it confirmed that the sofosbuvir-resistant NS5B substitution S282T was absent. This suggests the direct read translation approach implemented is of value for interpreting viral deep sequencing data.

Presence, persistence and effects of pre-treatment HIV-1 drug resistance variants detected using next generation sequencing: A Retrospective longitudinal study from rural coastal Kenya.

BACKGROUND: The epidemiology of HIV-1 drug resistance (HIVDR) determined by Sanger capillary sequencing, has been widely studied. However, much less is known about HIVDR detected using next generation sequencing (NGS) methods. We aimed to determine the presence, persistence and effect of pre-treatment HIVDR variants detected using NGS in HIV-1 infected antiretroviral treatment (ART) naïve participants from rural Coastal Kenya. METHODS: In a retrospective longitudinal study, samples from HIV-1 infected participants collected prior [n = 2 time-points] and after [n = 1 time-point] ART initiation were considered. An ultra-deep amplicon-based NGS assay, calling for nucleotide variants at >2.0% frequency of viral population, was used. Suspected virologic failure (sVF) was defined as a one-off HIV-1 viral load of >1000 copies/ml whilst on ART. RESULTS: Of the 50 eligible participants, 12 (24.0% [95% CI: 13.1-38.2]) had at least one detectable pre-treatment HIVDR variant against Protease Inhibitors (PIs, n = 6 [12%]), Nucleoside Reverse Transcriptase Inhibitors (NRTIs, n = 4 [8.0%]) and Non-NRTIs (n = 3 [6.0%]). Overall, 15 pre-treatment resistance variants were detected (frequency, range: 2.3-92.0%). A positive correlation was observed between mutation frequency and absolute load for NRTI and/or NNRTI variants (r = 0.761 [p = 0.028]), but not for PI variants (r = -0.117 [p = 0.803]). Participants with pre-treatment NRTI and/or NNRTI resistance had increased odds of sVF (OR = 6.0; 95% CI = 1.0-36.9; p = 0.054). CONCLUSIONS: Using NGS, pre-treatment resistance variants were common, though observed PI variants were unlikely transmitted, but rather probably generated de novo. Even when detected from a low frequency, pre-treatment NRTI and/or NNRTI resistance variants may adversely affect treatment outcomes.

Potential for diagnosis of infectious disease from the 100,000 Genomes Project Metagenomic Dataset: Recommendations for reporting results.

The identification of microbiological infection is usually a diagnostic investigation, a complex process that is firstly initiated by clinical suspicion. With the emergence of high-throughput sequencing (HTS) technologies, metagenomic analysis has unveiled the power to identify microbial DNA/RNA from a diverse range of clinical samples (1). Metagenomic analysis of whole human genomes at the clinical/research interface bypasses the steps of clinical scrutiny and targeted testing and has the potential to generate unexpected findings relating to infectious and sometimes transmissible disease. There is no doubt that microbial findings that may have a significant impact on a patient's treatment and their close contacts should be reported to those with clinical responsibility for the sample-donating patient. There are no clear recommendations on how such findings that are incidental, or outside the original investigation, should be handled. Here we aim to provide an informed protocol for the management of incidental microbial findings as part of the 100,000 Genomes Project which may have broader application in this emerging field. As with any other clinical information, we aim to prioritise the reporting of data that are most likely to be of benefit to the patient and their close contacts. We also set out to minimize risks, costs and potential anxiety associated with the reporting of results that are unlikely to be of clinical significance. Our recommendations aim to support the practice of microbial metagenomics by providing a simplified pathway that can be applied to reporting the identification of potential pathogens from metagenomic datasets. Given that the ambition for UK sequenced human genomes over the next 5 years has been set to reach 5 million and the field of metagenomics is rapidly evolving, the guidance will be regularly reviewed and will likely adapt over time as experience develops.

Determining the Origins of Human Immunodeficiency Virus Type 1 Drug-resistant Minority Variants in People Who Are Recently Infected Using Phylogenetic Reconstruction.

BACKGROUND: Drug-resistant minority variants (DRMinVs) detected in patients who recently acquired human immunodeficiency virus type 1 (HIV-1) can be transmitted, generated de novo through virus replication, or technical errors. The first form is likely to persist and result in treatment failure, while the latter two could be stochastic and transient. METHODS: Ultradeep sequencing of plasma samples from 835 individuals with recent HIV-1 infection in the United Kingdom was performed to detect DRMinVs at a mutation frequency between 2% and 20%. Sequence alignments including >110 000 HIV-1 partial pol consensus sequences from the UK HIV Drug Resistance Database (UK-HDRD), linked to epidemiological and clinical data from the HIV and AIDS Reporting System, were used for transmission cluster analysis. Transmission clusters were identified using Cluster Picker with a clade support of >90% and maximum genetic distances of 4.5% or 1.5%, the latter to limit detection to likely direct transmission events. RESULTS: Drug-resistant majority variants (DRMajVs) were detected in 66 (7.9%) and DRMinVs in 84 (10.1%) of the recently infected individuals. High levels of clustering to sequences in UK-HDRD were observed for both DRMajV (n = 48; 72.7%) and DRMinV (n = 63; 75.0%) sequences. Of these, 43 (65.2%) with DRMajVs were in a transmission cluster with sequences that harbored the same DR mutation compared to only 3 (3.6%) sequences with DRMinVs (P < .00001, Fisher exact test). Evidence of likely direct transmission of DRMajVs was observed for 25/66 (37.9%), whereas none were observed for the DRMinVs (P < .00001). CONCLUSIONS: Using a densely sampled HIV-infected population, we show no evidence of DRMinV transmission among recently infected individuals.

An Innovative Study Design to Assess the Community Effect of Interventions to Mitigate HIV Epidemics Using Transmission-Chain Phylodynamics.

Given globalization and other social phenomena, controlling the spread of infectious diseases has become an imperative public health priority. A plethora of interventions that in theory can mitigate the spread of pathogens have been proposed and applied. Evaluating the effectiveness of such interventions is costly and in many circumstances unrealistic. Most important, the community effect (i.e., the ability of the intervention to minimize the spread of the pathogen from people who received the intervention to other community members) can rarely be evaluated. Here we propose a study design that can build and evaluate evidence in support of the community effect of an intervention. The approach exploits molecular evolutionary dynamics of pathogens in order to track new infections as having arisen from either a control or an intervention group. It enables us to evaluate whether an intervention reduces the number and length of new transmission chains in comparison with a control condition, and thus lets us estimate the relative decrease in new infections in the community due to the intervention. We provide as an example one working scenario of a way the approach can be applied with a simulation study and associated power calculations.

Impact of Interferon-α Receptor-1 Promoter Polymorphisms on the Transcriptome of the Hepatitis B Virus-Associated Hepatocellular Carcinoma.

Background and aims: Genetic polymorphisms within the promoter of interferon-α receptor type-1 (IFNAR1) have been associated with the susceptibility to and the outcome of chronic hepatitis B virus (HBV) infection. However, the impact of these polymorphisms in the transcriptome of the HBV-associated hepatocellular carcinoma (HCC) remains largely unexplored. Methods: Using whole-genome and exome sequencing data from The Cancer Genome Atlas project, we characterized three single-nucleotide polymorphisms (SNPs: -568G/C, -408C/T, -3C/T) and one variable number tandem repeat [VNTR: -77(GT)n] within the IFNAR1 promoter sequence in 49 HCC patients. RNAseq data from 10 genotyped HCC samples were grouped according to their -77VNTR or -3SNP genotype to evaluate the impact of these polymorphisms on the differential expression on the HCC transcriptome. Results: There is a fourfold higher impact of the -77VNTR on the HCC transcriptome compared to the -3SNP (q < 0.1, p < 0.001). The expression of the primary IFNAR1 transcript is not affected by these polymorphisms but a secondary, HCC-specific transcript is expressed only in homozygous -77VNTR ≤8/≤8(GT)n samples (p < 0.05). At the same time, patients carrying at least one -77VNTR >8(GT) allele, presented a strong upregulation of the fibronectin-1 (FN-1) gene, which has been associated with the development of HCC. Gene Ontology and pathway enrichment analysis of the differentially expressed genes revealed a strong disruption of the PI3K-AKT signaling pathway, which can be partially triggered by the extracellular matrix FN-1. Conclusion: The IFNAR-1 promoter polymorphisms are not involved in the expression levels of the main IFNAR-1 transcript. The -77VNTR has a regulatory role on the expression of a secondary, truncated, HCC-specific transcript, which in turn coincides with disruptions in cancer-associated pathways and in FN-1 expression modifications.

Next generation sequencing of HIV-1 protease in the PIVOT trial of protease inhibitor monotherapy.

BACKGROUND: The PIVOT trial examined whether patients with suppressed viral load on combination antiretroviral therapy could be safely switched long-term to ritonavir-boosted protease inhibitor (PI) monotherapy. The main trial publication reported that only one of 296 patients allocated to PI monotherapy experienced a loss of drug options due to protease mutations (identified by local Sanger sequencing resistance tests) likely selected by study drug. OBJECTIVES: To assess if we had missed low frequency mutations, using a more sensitive methodology. STUDY DESIGN: We performed next generation sequencing (NGS) on all available frozen plasma samples with VL >1000 copies/ml from patients who were randomised to PI monotherapy. Assays were performed at Public Health England laboratories using a previously described method. Median coverage depth was 76,000 and the threshold for detection of minority variants was 2%. Drug susceptibility was predicted using the Stanford HIVdb algorithm. RESULTS: 17 of 26 potential samples, all from different patients, were identified and successfully tested. The median viral load was 6780 copies/ml and the median time since randomisation was 43 weeks. NGS revealed previously unidentified minority variant protease mutations (G73D, I54T, L89V) in three samples, at frequencies ranging between 2% and 10%. None of these mutations predicted intermediate or high level resistance, the trial primary outcome. DISCUSSION: This report adds to the body of evidence that ritonavir-boosted PI monotherapy, when used as a switch strategy with prompt detection of viral load rebound and early re-introduction of combination therapy, rarely leads to the development of clinically important protease resistance mutations.

Molecular epidemiology reveals the role of war in the spread of HIV in Ukraine.

Ukraine has one of the largest HIV epidemics in Europe, historically driven by people who inject drugs (PWID). The epidemic showed signs of stabilization in 2012, but the recent war in eastern Ukraine may be reigniting virus spread. We investigated the movement of HIV-infected people within Ukraine before and during the conflict. We analyzed HIV-1 subtype-A pol nucleotide sequences sampled during 2012-2015 from 427 patients of 24 regional AIDS centers and used phylogeographic analysis to reconstruct virus movement among different locations in Ukraine. We then tested for correlations between reported PWID behaviors and reconstructed patterns of virus spread. Our analyses suggest that Donetsk and Lugansk, two cities not controlled by the Ukrainian government in eastern Ukraine, were significant exporters of the virus to the rest of the country. Additional analyses showed that viral dissemination within the country changed after 2013. Spearman correlation analysis showed that incoming virus flow was correlated with the number of HIV-infected internally displaced people. Additionally, there was a correlation between more intensive virus movement and locations with a higher proportion of PWID practicing risky sexual behaviors. Our findings suggest that effective prevention responses should involve internally displaced people and people who frequently travel to war-affected regions. Scale-up of harm reduction services for PWID will be an important factor in preventing new local HIV outbreaks in Ukraine.

A European multi-centre External Quality Assessment (EQA) study on phenotypic and genotypic methods used for Herpes Simplex Virus (HSV) drug resistance testing.

BACKGROUND: Herpes Simplex Virus (HSV) drug resistance is a significant public health concern among immunocompromised individuals. Phenotypic assays are considered the gold standard method for detecting HSV drug resistance. However, plaque reduction assays (PRAs) are technically demanding, often with long turnaround times of up to four weeks. In contrast, genotypic tests can be performed within a few days. OBJECTIVES: The development and coordination of the first European External Quality Assessment (EQA) study to evaluate phenotypic and genotypic methods used for HSV drug resistance testing in specialised reference laboratories. STUDY DESIGN: Four HSV-1 or HSV-2 strains with different antiviral susceptibility profiles were isolated from clinical samples. Isolates were quantified by qPCR, and aliquoted in culture medium. One isolate was distributed at two dilutions to help assess assay sensitivity. The panel was distributed to five European centres with a six-week deadline for the return of phenotypic and genotypic results, together with clinical reports. RESULTS: Four out of five participating labs returned results by the deadline. Limited results were later available from the fifth lab. Phenotypic and genotypic data were largely, but not completely, concordant. An unusual resistance profile shown by one of the samples was explained by the detection of a mixed virus population after extensive further investigation by one of the centres. CONCLUSIONS: Discordant clinical outputs reflecting the diversity of phenotypic methodologies demonstrated the utility of this exercise. With emerging genotypic technologies looking to supplant phenotyping, there is a need for curated public databases, accessible interpretation tools and standardised control materials for quality management. By establishing a network of testing laboratories, we hope that this EQA scheme will facilitate ongoing progress in this area.

Efficient and unbiased metagenomic recovery of RNA virus genomes from human plasma samples.

RNA viruses cause significant human pathology and are responsible for the majority of emerging zoonoses. Mainstream diagnostic assays are challenged by their intrinsic diversity, leading to false negatives and incomplete characterisation. New sequencing techniques are expanding our ability to agnostically interrogate nucleic acids within diverse sample types, but in the clinical setting are limited by overwhelming host material and ultra-low target frequency. Through selective host RNA depletion and compensatory protocol adjustments for ultra-low RNA inputs, we are able to detect three major blood-borne RNA viruses - HIV, HCV and HEV. We recovered complete genomes and up to 43% of the genome from samples with viral loads of 104 and 103 IU/ml respectively. Additionally, we demonstrated the utility of this method in detecting and characterising members of diverse RNA virus families within a human plasma background, some present at very low levels. By applying this method to a patient sample series, we have simultaneously determined the full genome of both a novel subtype of HCV genotype 6, and a co-infecting human pegivirus. This method builds upon earlier RNA metagenomic techniques and can play an important role in the surveillance and diagnostics of blood-borne viruses.