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.

In patients failing cART with LLV, HIV-1 genotyping provides reliable and reproducible results that are informative about emerging drug resistance.

in this work we present easyprimer, a user-friendly online tool developed to assist pan-pcR and High Resolution Melting (HRM) primer design. The tool finds the most suitable regions for primer design in a gene alignment and returns a clear graphical representation of their positions on the consensus sequence. EasyPrimer is particularly useful in difficult contexts, e.g. on gene alignments of hundreds of sequences and/or on highly variable genes. HRM analysis is an emerging method for fast and cost saving bacterial typing and an HRM scheme of six primer pairs on five Multi-Locus Sequence Type (MLST) genes is already available for Klebsiella pneumoniae. We validated the tool designing a scheme of two HRM primer pairs on the hypervariable gene wzi of Klebsiella pneumoniae and compared the two schemes. the wzi scheme resulted to have a discriminatory power comparable to the HRM MLST scheme, using only one third of primer pairs. then we successfully used the wzi HRM primer scheme to reconstruct a Klebsiella pneumoniae nosocomial outbreak in few hours. the use of hypervariable genes reduces the number of HRM primer pairs required for bacterial typing allowing to perform cost saving, large-scale surveillance programs.Most methods used for the identification and typing of prokaryotes are based on DNA amplification and sequencing. Indeed, the sequence of specific genes can harbour enough information to classify bacteria at species, subspecies or also to a clonal level. For instance, Multi-Locus Sequence Typing (MLST) is one of the most used methods for bacterial typing and it is based on the amplification and sequencing of few housekeeping genes 1 . During the last ten years, the analysis of the entire bacterial genome by Whole Genome Sequencing (WGS) approach revolutionized the field, drastically increasing the typing precision 1 .The reconstruction of nosocomial outbreaks is one of the most important clinical applications of bacterial typing. A nosocomial outbreak occurs when the number of patients infected by a pathogen increases above the expected in a limited time 2 . In these situations, it is fundamental to determine the clonality of bacteria causing disease in the patients to define the proper strategy to handle the emergency. Pulsed-Field Gel Electrophoresis (PFGE), MLST and WGS are the most frequently applied molecular methods in outbreak investigation 1 .During a nosocomial outbreak, clinicians need bacterial typing information in the shortest time possible. Despite the high potential of WGS in outbreak reconstruction, the sequencing of a complete genome requires two to four working days, introducing an important time lag. Similarly, PFGE typing requires five days and also MLST needs few days. During the last years, the High Resolution Melting (HRM) assay has emerged as a low-cost and fast method for bacterial typing, particularly promising for epidemiological applications 3-6 . HRM is a single-step

BackgroundHBsAg immune-escape mutations can favor HBV-transmission also in vaccinated individuals, promote immunosuppression-driven HBV-reactivation, and increase fitness of drug-resistant strains. Stop-codons can enhance HBV oncogenic-properties. Furthermore, as a consequence of the overlapping structure of HBV genome, some immune-escape mutations or stop-codons in HBsAg can derive from drug-resistance mutations in RT. This study is aimed at gaining insight in prevalence and characteristics of immune-associated escape mutations, and stop-codons in HBsAg in chronically HBV-infected patients experiencing nucleos(t)ide analogues (NA) in Europe.MethodsThis study analyzed 828 chronically HBV-infected European patients exposed to ≥ 1 NA, with detectable HBV-DNA and with an available HBsAg-sequence.The immune-associated escape mutations and the NA-induced immune-escape mutations sI195M, sI196S, and sE164D (resulting from drug-resistance mutation rtM204 V, rtM204I, and rtV173L) were retrieved from literature and examined. Mutations were defined as an aminoacid substitution with respect to a genotype A or D reference sequence.ResultsAt least one immune-associated escape mutation was detected in 22.1% of patients with rising temporal-trend. By multivariable-analysis, genotype-D correlated with higher selection of ≥ 1 immune-associated escape mutation (OR[95%CI]:2.20[1.32–3.67], P = 0.002). In genotype-D, the presence of ≥ 1 immune-associated escape mutations was significantly higher in drug-exposed patients with drug-resistant strains than with wild-type virus (29.5% vs 20.3% P = 0.012). Result confirmed by analysing drug-naïve patients (29.5% vs 21.2%, P = 0.032). Strong correlation was observed between sP120T and rtM204I/V (P < 0.001), and their co-presence determined an increased HBV-DNA.At least one NA-induced immune-escape mutation occurred in 28.6% of patients, and their selection correlated with genotype-A (OR[95%CI]:2.03[1.32–3.10],P = 0.001).Finally, stop-codons are present in 8.4% of patients also at HBsAg-positions 172 and 182, described to enhance viral oncogenic-properties.ConclusionsImmune-escape mutations and stop-codons develop in a large fraction of NA-exposed patients from Europe. This may represent a potential threat for horizontal and vertical HBV transmission also to vaccinated persons, and fuel drug-resistance emergence.Electronic supplementary materialThe online version of this article (10.1186/s12879-018-3161-2) contains supplementary material, which is available to authorized users.

vis, et al.. Rare occurrence of doravirine resistance-associated mutations in HIV-1-infected treatmentnaive patients. 27 Salpêtrière 45-83 Bd de l'hôpital 75013 Paris, France. Phone: 33 1 42 17 58 42. Fax: 33 1 42 28 17 74 11. ABSTRACT 34 35 Background: Doravirine is a novel HIV-1 NNRTIs recently shown to be non-inferior both to 36 darunavir/ritonavir and efavirenz in combination therapy with two nucleoside reverse 37 transcriptase inhibitor in treatment-naïve patients. Doravirine has an in vitro resistance profile 38 that is distinct from other NNRTIs and retains activity against viruses containing the most 39 frequently transmitted NNRTIs mutations. The aim of this study was to examine the 40 prevalence of doravirine associated mutations in HIV-1-infected treatment-naïve patients in 41 Europe. 42 Patients and methods: From 2010 to 2016, 9764 treatment-naïve patients were tested for 43 NNRTIs antiretroviral drug resistance by bulk sequencing in Greece, Italy and France. We 44 studied the prevalence of doravirine resistance associated mutations previously identified in 45 vitro: V106A/M, V108I, Y188L, V190S, H221Y, F227C/L/V, M230I/L, L234I, P236L, 46 Y318F and K103N/Y181C. 47 107 comparisons were carried out with Fisher's exact test using the BiostatTGV web site 108 (https://biostatgv.sentiweb.fr/?module=tests).109 110 RESULTS 111 112 Distribution of HIV-1 subtypes in antiretroviral-naïve patients 113 A total a 9764 RT sequences obtained between 2010 and 2016 for HIV-1 treatment-naïve 114 patients in routine clinical care were analyzed (