The Ebola virus disease (EVD) epidemic in West Africa is the largest on record, responsible for >28,599 cases and >11,299 deaths 1. Genome sequencing in viral outbreaks is desirable in order to characterize the infectious agent to determine its evolutionary rate, signatures of host adaptation, identification and monitoring of diagnostic targets and responses to vaccines and treatments. The Ebola virus genome (EBOV) substitution rate in the Makona strain has been estimated at between 0.87 × 10−3 to 1.42 × 10−3 mutations per site per year. This is equivalent to 16 to 27 mutations in each genome, meaning that sequences diverge rapidly enough to identify distinct sub-lineages during a prolonged epidemic 2-7. Genome sequencing provides a high-resolution view of pathogen evolution and is increasingly sought-after for outbreak surveillance. Sequence data may be used to guide control measures, but only if the results are generated quickly enough to inform interventions 8. Genomic surveillance during the epidemic has been sporadic due to a lack of local sequencing capacity coupled with practical difficulties transporting samples to remote sequencing facilities 9. In order to address this problem, we devised a genomic surveillance system that utilizes a novel nanopore DNA sequencing instrument. In April 2015 this system was transported in standard airline luggage to Guinea and used for real-time genomic surveillance of the ongoing epidemic. Here we present sequence data and analysis of 142 Ebola virus (EBOV) samples collected during the period March to October 2015. We were able to generate results in less than 24 hours after receiving an Ebola positive sample, with the sequencing process taking as little as 15-60 minutes. We show that real-time genomic surveillance is possible in resource-limited settings and can be established rapidly to monitor outbreaks.

Table of Contents 1. Levels of evidence1.1 Reference2. Introduction3. Auditable targets4. Table summaries4.1 Initial diagnosis4.2 Assessment of ART‐naïve individuals4.3 ART initiation4.4 Initial assessment following commencement of ART4.5 Routine monitoring on ART4.6 References5. Newly diagnosed and transferring HIV‐positive individuals5.1 Initial HIV‐1 diagnosis5.2 Tests to determine whether acquisition of HIV infection is recent5.3 Individuals transferring care from a different HIV healthcare setting5.4 Communication with general practitioners and shared care5.5 Recommendations5.6 References6. Patient history6.1 Initial HIV‐1 diagnosis6.2 Monitoring of ART‐naïve patients6.3 Pre‐ART initiation assessment6.4 Monitoring individuals established on ART6.5 Assessment of adherence6.6 Recommendations6.7 References7. Examination7.1 Recommendations8. Identifying the need for psychological support8.1 References9. Assessment of immune status9.1 CD4 T cell counts9.2 CD4 T cell percentage9.3 References10. HIV viral load10.1 Initial diagnosis/ART naïve10.2 Post ART initiation10.3 Individuals established on ART10.4 Recommendations10.5 References11. Technical aspects of viral load testing11.1 References12. Viral load kinetics during ART and viral load ‘blips’12.1 References13. Proviral DNA load13.1 References14. Resistance testing14.1 Initial HIV‐1 diagnosis14.2 ART‐naïve14.3 Post treatment initiation14.4 ART‐experienced14.5 References15. Subtype determination15.1 Disease progression15.2 Transmission15.3 Performance of molecular diagnostic assays15.4 Response to therapy15.5 Development of drug resistance15.6 References16. Other tests to guide use of specific antiretroviral agents16.1 Tropism testing16.2 HLA B*5701 testing16.3 References17. Therapeutic drug monitoring17.1 Recommendations17.2 References18. Biochemistry testing18.1 Introduction18.2 Liver function18.3 Renal function18.4 Dyslipidaemia in HIV‐infected individuals18.5 Other biomarkers18.6 Bone disease in HIV‐infected patients18.7 References19. Haematology19.1 Haematological assessment and monitoring19.2 Recommendations19.3 References20. Serology20.1 Overview20.2 Hepatitis viruses20.3 Herpes viruses20.4 Measles and rubella20.5 Cytomegalovirus (CMV)20.6 References21. Other microbiological screening21.1 Tuberculosis screening21.2 Toxoplasma serology21.3 Tropical screening21.4 References22. Sexual health screening including anal and cervical cytology22.1 Sexual history taking, counselling and sexually transmitted infection (STI) screening22.2 Cervical and anal cytology22.3 Recommendations22.4 References23. Routine monitoring recommended for specific patient groups23.1 Women23.2 Older age23.3 Injecting drug users23.4 Individuals coinfected with HBV and HCV23.5 Late presenters23.6 References Appendix

The objective of this study was to track the evolution of sequence changes in both the heptad region 1 (HR1) and HR2 domains of gp41 associated with resistance to enfuvirtide (ENF) in a patient cohort receiving longterm ENF treatment. We studied 17 highly antiretroviral agent-experienced patients receiving long-term ENF treatment with virological rebound or a lack of suppression. Sixty-two samples obtained after between 5 and 107 weeks of ENF therapy were analyzed. Baseline samples from 15 of these 17 patients were available for analysis. Viruses from five samples from four patients were also sequenced after the cessation of ENF therapy. Drug susceptibilities were assessed by a pseudotype virus reporter assay. We identified HR1 and HR2 sequence changes over time in relation to the baseline sequences. Mutations in HR1 (amino acids 36 to 45) were noted in all cases, including previously unreported changes N42Q/H and N43Q. In addition to a range of HR2 sequence changes at polymorphic sites, isolates from 6 of 17 (35%) patients developed an S138A substitution in the HR2 domain at least 8 weeks after the start of ENF treatment and also subsequent to the first emergence of HR1 mutations. In most, but not all, cases the S138A mutation accompanied HR1 mutations at position 43. Molecular modeling demonstrates the close proximity of S138A with amino acids 40 and 45 in HR1. Of note, isolates in samples available from four patients demonstrated the loss of both the HR1 and the S138A HR2 mutations following the cessation of therapy. We show that the S138A HR2 mutation increased the level of resistance by approximately threefold over that conferred by the HR1 mutation N43D. Continual evolution of HR1 in the domain from amino acids 36 to 45 was observed during long-term ENF therapy. We have identified, for the first time, an ENF resistance-associated HR2 mutation, S138A, which appeared in isolates from 6 of 17 patients with virological failure and demonstrated its potential to contribute to drug resistance. We propose that this represents a possible secondary and/or compensatory mutation, particularly when it coexists with mutations at position 43 in HR-1.The envelope glycoprotein (env) of human immunodeficiency virus type 1 (HIV-1) plays a key role in viral entry into the host cell (11, 31). Env contains two noncovalently associated subunits, gp120 and gp41. The binding of gp120 to the cellular receptor CD4 and a chemokine coreceptor triggers a series of complex conformational changes in gp41 that lead to the fusion of viral and cellular membranes. There are three functional regions in the ectodomain of gp41: the fusion peptide, heptad repeat 1 (HR1) and HR2, and the transmembrane region. Crystallographic studies have demonstrated that the fusion-active (fusogenic) conformation of gp41 is a six-helix bundle structure in which three N-terminal helices (HR1) form a central trimeric coiled coil and three C-terminal helices (HR2) pack in an antiparallel manner into hydrophobic grooves of the coiled core. The hairpin interactions ...

SummaryBackgroundAntiretroviral therapy (ART) is crucial for controlling HIV-1 infection through wide-scale treatment as prevention and pre-exposure prophylaxis (PrEP). Potent tenofovir disoproxil fumarate-containing regimens are increasingly used to treat and prevent HIV, although few data exist for frequency and risk factors of acquired drug resistance in regions hardest hit by the HIV pandemic. We aimed to do a global assessment of drug resistance after virological failure with first-line tenofovir-containing ART.MethodsThe TenoRes collaboration comprises adult HIV treatment cohorts and clinical trials of HIV drug resistance testing in Europe, Latin and North America, sub-Saharan Africa, and Asia. We extracted and harmonised data for patients undergoing genotypic resistance testing after virological failure with a first-line regimen containing tenofovir plus a cytosine analogue (lamivudine or emtricitabine) plus a non-nucleotide reverse-transcriptase inhibitor (NNRTI; efavirenz or nevirapine). We used an individual participant-level meta-analysis and multiple logistic regression to identify covariates associated with drug resistance. Our primary outcome was tenofovir resistance, defined as presence of K65R/N or K70E/G/Q mutations in the reverse transcriptase (RT) gene.FindingsWe included 1926 patients from 36 countries with treatment failure between 1998 and 2015. Prevalence of tenofovir resistance was highest in sub-Saharan Africa (370/654 [57%]). Pre-ART CD4 cell count was the covariate most strongly associated with the development of tenofovir resistance (odds ratio [OR] 1·50, 95% CI 1·27–1·77 for CD4 cell count <100 cells per μL). Use of lamivudine versus emtricitabine increased the risk of tenofovir resistance across regions (OR 1·48, 95% CI 1·20–1·82). Of 700 individuals with tenofovir resistance, 578 (83%) had cytosine analogue resistance (M184V/I mutation), 543 (78%) had major NNRTI resistance, and 457 (65%) had both. The mean plasma viral load at virological failure was similar in individuals with and without tenofovir resistance (145 700 copies per mL [SE 12 480] versus 133 900 copies per mL [SE 16 650; p=0·626]).InterpretationWe recorded drug resistance in a high proportion of patients after virological failure on a tenofovir-containing first-line regimen across low-income and middle-income regions. Effective surveillance for transmission of drug resistance is crucial.FundingThe Wellcome Trust.

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