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Although antiretroviral therapy (ART) is effective in suppressing viral replication, HIV-1 persists in reservoirs and rebounds after ART has been stopped. However, a very few people (eg, elite and post-treatment controllers) are able to maintain viral loads below detection limits without ART, constituting a realistic model for long-term HIV remission. Here, we describe the HIV control mechanisms of an individual who showed exceptional post-treatment control for longer than 15 years.We report the case of a Hispanic woman aged 59 years with sexually acquired acute HIV infection, who was included in an immune-mediated primary HIV infection trial involving a short course of ciclosporine A, interleukin-2, granulocyte macrophage colony-stimulating factor, and pegylated interferon alfa, followed by analytical treatment interruption. We did the following viral assays: total and integrated HIV-1 DNA in CD4 T cells and rectal tissue, quantitative viral outgrowth assay, HIV-1 infectivity in peripheral blood mononuclear cells and CD4 T-cell cultures and viral inhibitory activity by natural killer (NK) and CD8 T cells. NK and T-cell phenotypes were determined by flow cytometry. HLA, killer cell immunoglobulin-like receptors, Δ32CCR5, and NKG2C alleles were genotyped.After ART and immunomodulatory treatment, the person maintained undetectable plasma viral load for 15 years. HIV-1 subtype was CFR_02AG, CCR5-tropic. We found progressive reductions in viral reservoir during the 15-year treatment interruption: total HIV DNA (from 4573·50 copies per 10We described long-term remission in a woman aged 59 years who was treated during primary HIV infection and has maintained undetectable viral load for 15 years without ART. Replication-competent HIV-1 was isolated. NKG2C-memory-like NK cells and γδ T cells were associated with the control viral replication. Strategies promoting these cells could bring about long-term HIV remission.Fondo Europeo para el Desarrollo Regional (FEDER), SPANISH AIDS Research Network (RIS), Fondo de Investigación Sanitaria (FIS), HIVACAT, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CERCA Programme/Generalitat de Catalunya, la Caixa Foundation, and Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC).For the Spanish translation of the abstract see Supplementary Materials section.

Bronchoscopy is a widely use technique in critically ill patients. Nosocomial coinfections are a cause of morbidity and mortality in intensive care units.Our aim was to describe bronchoscopy findings and analyze microbiological profile and probably coinfection through bronchial aspirate (BA) samples in patients with coronavirus disease 2019 pneumonia requiring intensive care unit admission.Retrospective observational study analyzing the BA samples collected from intubated patients with coronavirus disease 2019 in a referral Hospital (Spain).One hundred fifty-five consecutive BA samples were collected from 75 patients. Ninety (58%) were positive cultures for different microorganisms, 11 (7.1%) were polymicrobial, and 37 (23.7%) contained resistant microorganisms. There was a statistically significant association between increased days of orotracheal intubation and positive BA (18.9 vs. 10.9 d, P0.01), polymicrobial infection (22.11 vs. 13.54, P0.01) and isolation of resistant microorganisms (18.88 vs. 10.94, P0.01). In 88% of the cases a new antibiotic or change in antibiotic treatment was made.Bronchoscopy in critically ill patient was safe and could be useful to manage these patients and conduct the microbiological study, that seems to be higher and different than in nonepidemic periods. The longer the intubation period, the greater the probability of coinfection, isolation of resistant microorganisms and polymicrobial infection.

BackgroundTransmission of resistance mutations to integrase strand transfer inhibitors (INSTIs) in HIV-infected patients may compromise the efficacy of first-line antiretroviral regimens currently recommended worldwide. Continued surveillance of transmitted drug resistance (TDR) is thus warranted.ObjectivesWe evaluated the rates and effects on virological outcomes of TDR in a 96 week prospective multicentre cohort study of ART-naive HIV-1-infected subjects initiating INSTI-based ART in Spain between April 2015 and December 2016.MethodsPre-ART plasma samples were genotyped for integrase, protease and reverse transcriptase resistance using Sanger population sequencing or MiSeq™ using a ≥ 20% mutant sensitivity cut-off. Those present at 1%–19% of the virus population were considered to be low-frequency variants.ResultsFrom a total of 214 available samples, 173 (80.8%), 210 (98.1%) and 214 (100.0%) were successfully amplified for integrase, reverse transcriptase and protease genes, respectively. Using a Sanger-like cut-off, the overall prevalence of any TDR, INSTI-, NRTI-, NNRTI- and protease inhibitor (PI)-associated mutations was 13.1%, 1.7%, 3.8%, 7.1% and 0.9%, respectively. Only three (1.7%) subjects had INSTI TDR (R263K, E138K and G163R), while minority variants with integrase TDR were detected in 9.6% of subjects. There were no virological failures during 96 weeks of follow-up in subjects harbouring TDR as majority variants.ConclusionsTransmitted INSTI resistance remains rare in Spain and, to date, is not associated with virological failure to first-line INSTI-based regimens.

We report proof-of-principle experiments regarding a dynamic microarray protocol enabling accurate and semi-quantitative DNA analysis for re-sequencing, fingerprinting and genotyping. Single-stranded target molecules hybridise to surface-bound probes during initial gradual cooling with high-fidelity. Real-time tracking of target denaturation (via fluorescence) during a 'dynamic' gradual heating phase permits 'melt-curve' analysis. The probe most closely matching the target sequence is identified based on the highest melting temperature. We demonstrated a >99% re-sequencing accuracy and a potential detection rate of 1% for SNPs. Experiments employing Hypericum ribosomal ITS regions and HIV genomes illustrated a reliable detection level of 5% plus simultaneous re-sequencing and genotyping. Such performance suggests a range of potential real-world applications involving rapid sequence interrogation, for example, in the Covid-19 pandemic. Guidance is offered towards the development of a commercial platform and dedicated software required to bring this technique into mainstream science.

BackgroundNosocomial co-infections are a cause of morbidity and mortality in Intensive Care Units (ICU).ObjectivesOur aim was to describe bronchoscopy findings and analyse co-infection through bronchial aspirate (BA) samples in patients with COVID-19 pneumonia requiring ICU admission.MethodsWe conducted a retrospective observational study, analysing the BA samples collected from intubated patients with COVID-19 to diagnose nosocomial respiratory infection.ResultsOne-hundred and fifty-five consecutive BA samples were collected from 75 patients. Of them, 90 (58%) were positive cultures for different microorganisms, 11 (7.1%) were polymicrobial, and 37 (23.7%) contained resistant microorganisms. There was a statistically significant association between increased days of orotracheal intubation (OTI) and positive BA (18.9 days versus 10.9 days, pConclusionNosocomial respiratory infection in intubated COVID-19 patients seems to be higher than in non-epidemic periods. The longer the intubation period, the greater the probability of co-infection, isolation of resistant microorganisms and polymicrobial infection. Microbiological sampling through BA is an essential tool to manage these patients appropriately.

Background In rhesus macaques, simian immunodeficiency virus infection is followed by expansion of enteric viruses but has a limited impact on the gut bacteriome. To understand the longitudinal effects of HIV-1 infection on the human gut microbiota, we prospectively followed 49 Mozambican subjects diagnosed with recent HIV-1 infection (RHI) and 54 HIV-1-negative controls for 9–18 months and compared them with 98 chronically HIV-1-infected subjects treated with antiretrovirals (n = 27) or not (n = 71). Results We show that RHI is followed by increased fecal adenovirus shedding, which persists during chronic HIV-1 infection and does not resolve with ART. Recent HIV-1 infection is also followed by transient non-HIV-specific changes in the gut bacterial richness and composition. Despite early resilience to change, an HIV-1-specific signature in the gut bacteriome—featuring depletion of Akkermansia, Anaerovibrio, Bifidobacterium, and Clostridium—previously associated with chronic inflammation, CD8+ T cell anergy, and metabolic disorders, can be eventually identified in chronically HIV-1-infected subjects. Conclusions Recent HIV-1 infection is associated with increased fecal shedding of eukaryotic viruses, transient loss of bacterial taxonomic richness, and long-term reductions in microbial gene richness. An HIV-1-associated microbiome signature only becomes evident in chronically HIV-1-infected subjects. Electronic supplementary material The online version of this article (10.1186/s40168-019-0687-5) contains supplementary material, which is available to authorized users.

Human immunodeficiency virus (HIV)-1 infection causes severe gut and systemic immune damage, but its effects on the gut microbiome remain unclear. Previous shotgun metagenomic studies in HIV-negative subjects linked low-microbial gene counts (LGC) to gut dysbiosis in diseases featuring intestinal inflammation. Using a similar approach in 156 subjects with different HIV-1 phenotypes, we found a strong, independent, dose-effect association between nadir CD4+ T-cell counts and LGC. As in other diseases involving intestinal inflammation, the gut microbiomes of subjects with LGC were enriched in gram-negative Bacteroides, acetogenic bacteria and Proteobacteria, which are able to metabolize reactive oxygen and nitrogen species; and were depleted in oxygen-sensitive methanogenic archaea and sulfate-reducing bacteria. Interestingly, subjects with LGC also showed increased butyrate levels in direct fecal measurements, consistent with enrichment in Roseburia intestinalis despite reductions in other butyrate producers. The microbiomes of subjects with LGC were also enriched in bacterial virulence factors, as well as in genes associated with beta-lactam, lincosamide, tetracycline, and macrolide resistance. Thus, low nadir CD4+ T-cell counts, rather than HIV-1 serostatus per se, predict the presence of gut dysbiosis in HIV-1 infected subjects. Such dysbiosis does not display obvious HIV-specific features; instead, it shares many similarities with other diseases featuring gut inflammation. Fundació Glòria Soler Fundació Catalunya-La Pedrera Gala SIDA 2015-2016 Nit per la Recerca a la Catalunya Central 2015 edition People in Red-Barcelona 2016 edition RED de SIDA RD16/0025/0041 ISCIII European Regional Develpment Fund (ERDF) Agencia de Gestio d´Ajuts Universitaris i de Recerca (AGAUR) Secretaria d´Universitats i Recerca del Departament d´Economia i Coneixement de la Generalitat de Catalunya Ministerio de Economia y Competitividad. España Universidad de Whashington

The impact of host genetics on gut microbial dynamics is debated. No study to date has investigated the possible role of host genetics in shaping the gut microbiota in HIV-1 infected subjects. With the aim of generating preliminary data to inform future host genetic studies, we performed an exploratory host exome analysis of 147 subjects either infected or at risk of becoming infected with HIV-1 from the MetaHIV cohort in Barcelona. Using a DNA microarray chip, we sought to identify host genetic variants associated to three specific microbial features with a potentially inheritable component, and which were previously found to be associated with gut dysbiosis in HIV infection, i.e.: gut enterotype, presence of methanogenic archaea and microbial gene richness. After correction for multiple comparisons, we did not observe any statistically significant association between the host’s genetic landscape and the explored gut microbiome traits. These findings will help design future, adequately-powered studies to assess the influence of host genetics in the microbiome of HIV-1-infected subjects.

No controlled comparisons between dolutegravir/lamivudine or dolutegravir maintenance therapy have been done. We hypothesized that these options would have similar efficacy to triple ART.We used an open-label non-inferiority randomized controlled trial comprising two phases: phase A was established to test that experimental arms did not have an unacceptable (≥5%) failure rate; phase B was intended to include the full number of patients followed for 48 weeks. Treated HIV-1-infected adults with viral load50 copies/mL for ≥12 months, no prior viral failure or resistance mutations to study drugs, nadir CD4200 cells/mm3, and hepatitis B virus surface antigen negative were randomized 1:1:1 to maintain triple therapy (control arm), or to switch to dolutegravir/lamivudine, or to dolutegravir monotherapy stratifying by anchor drug. Premature discontinuation was considered if viral failure or therapy interruption due to adverse events, concurrent illness, protocol deviation or patient's wish occurred. Blips were registered. Planned phase A results at 24 weeks are reported here. The study is registered at EudraCT: 201500027435.Ninety-one (control, n = 31; dual therapy, n = 29; monotherapy, n = 31) patients were randomized. Three patients (none previously exposed to integrase inhibitors) prematurely discontinued treatment due to viral failure: dolutegravir/lamivudine (n = 1), no resistance mutations (subject A); dolutegravir (n = 2), N155H, S147G and Q148R resistance mutations (subject B), and E138K, G140S and N155H resistance mutations (subject C). There were no discontinuations for other reasons. One patient (dolutegravir/lamivudine) experienced a blip in viral load. The Data Safety Monitoring Board recommended stopping the dolutegravir monotherapy arm.In contrast to dolutegravir/lamivudine, a higher than expected risk of viral failure with development of cross-resistance integrase mutations occurred with dolutegravir maintenance monotherapy.

Objectives To investigate the prevalence and patterns of major and accessory resistance mutations associated with integrase strand transfer inhibitors (INSTIs), across diverse HIV-1 subtypes in sub-Saharan Africa. Methods pol gene sequences were obtained using Illumina next-generation sequencing from 425 INSTI-naive HIV-infected adults from Kenya (21.2%), Nigeria (7.3%), South Africa (22.8%), Uganda (25.2%) and Zambia (23.5%). Drug resistance interpretation was based on the IAS 2017 mutation list and accessory mutations from Stanford HIVdb with resistance penalty scores of ≥10 to at least 1 INSTI. Resistance was further classified based on sensitivity thresholds of ≥20% (Sanger sequencing) and 1%–20% for low-frequency variants (next-generation sequencing). Results Of 425 genotypes, 48.7% were subtype C, 28.5% A, 10.1% D, 2.8% G and 9.9% were recombinants. Major INSTI resistance mutations were detected only at Conclusions Major INSTI resistance mutations were rare and only occurred at low-level resistance detection thresholds. INSTI-based regimens are expected to be effective across the different major HIV-1 subtypes in the region.

Preventive HIV-1 vaccine strategies rely on the elicitation of broadly neutralizing antibody (bNAb) responses, but their induction in vivo by vaccination remains challenging. Considering that the ability of an epitope to elicit effective humoral immunity depends on its exposure on the virion, we have used a reverse genetics approach to select variants from an HIV-1 AC10_29 randomly mutated envelope library that showed increased affinity for a selected bNAb (4E10 bNAb targeting the HIV-1 MPER region). Isolated envelope sequences were analyzed by deep-sequencing showing a small number of dominant changes, including the loss of four potential N-linked glycosylation sites and disruption of the V1/V2 loop. Accordingly, the dominant variant (LR1-C1), showed not only increased affinity for MPER bNAbs 4E10 and 2F5, but also higher affinity for an additional antibody targeting the V3 loop (447-52D) that could be a consequence of an open conformation tier 1-like Env. Furthermore, the amino acids specific for the selected variant are associated with an increased sensitivity for 4E10 and 2F5 antibodies. In vivo studies showed that sera from mice immunized with LR1-C1 viruses possessed an improved neutralizing activity compared to the wild-type AC10_29 env. While Virus Like Particles (VLPs) carrying this envelope were unable to induce detectable neutralizing activity in immunized rabbits, one animal showed antibody response to the 4E10-proximal region. Our data establish a novel approach that has the potential to yield HIV envelope immunogen sequences that direct antibody responses to specific envelope regions. © 2018 Beltran-Pavez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited., The authors do not have any conflict of interests. We wish to thank F. Diez-Fuertes from the AIDS Immunopathology Unit (Instituto de Salud Carlos III, Centro Nacional de Microbiología, Madrid, Spain) for help with the statistical analysis, Dr. R. Wagner from the Institute of Medical Microbiology and Hygiene, Molecular Microbiology (Virology), University Regensburg (Regensburg, Germany) for 96ZM 651_HXB2_V3 SOSIP, Dr. Q. Sattentau from The Sir William Dunn School of Pathology, University of Oxford (Oxford, U. K.) for PGT151 antibody and pNL4-3 plasmid and antibodies 4E10, 2F5, 447-52D, VRC01, 5F3, b12, PG16 and 2G12 were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH.

BACKGROUND: Implementation of ultrasensitive HIV drug resistance tests for routine clinical use is hampered by uncertainty about the clinical relevance of drug-resistant minority variants. We assessed different detection thresholds for pretreatment drug resistance to predict an increased risk of virological failure.METHODS: We did a case-control study nested within a prospective multicountry cohort. Our study included patients from 12 clinical sites in Kenya, Nigeria, South Africa, Uganda, and Zambia. We defined cases as patients with virological failure (ie, those who had either viral load ≥400 copies per mL at 12 months or had switched to second-line antiretroviral therapy [ART] as a result of virological failure before 12 months) and controls as those with viral suppression (viral load FINDINGS: Paired viral load results before ART and at month 12 of follow-up were available from 1896 participants. We identified 178 patients with virological failure and selected 338 matched controls. We excluded 117 patients from pretreatment drug resistance analysis; therefore, 152 cases of virological failure and 247 controls were included in the final analysis. With the IAS-USA mutation list, at a detection threshold of 20% or more in patients with pretreatment drug resistance, the adjusted odds ratio (OR) for virological failure was 9·2 (95% CI 4·2-20·1) compared with those without pretreatment drug resistance. Lowering the threshold resulted in adjusted ORs of virological failure of 6·8 (95% CI 3·3-13·9) at the 10% threshold, 7·6 (3·4-17·1) at the 5% threshold, and 4·5 (2·0-10·2) at the 1% threshold. Lowering the detection threshold from 20% improved the sensitivity (ie, ability to identify cases) from 12% (n=18) to 13% (n=19) at detection threshold 10%, to 15% (n=23) at detection threshold 5%, and to 17% (n=26) at detection threshold 1%, but caused a slight reduction in specificity (ie, ability to identify controls) from 98% (n=241) to 96% (n=238) at the 10% threshold, 96% (n=236) at the 5% threshold, and a larger reduction to 92% (n=227) at the 1% threshold. Diagnostic ORs were 5·4 (95% CI 2·1-13·9) at the 20% threshold, 3·8 (1·7-8·6) at the 10% threshold, 3·8 (1·8-8·1) at the 5% threshold, and 2·3 (1·2-4·2) at the 1% threshold. Use of the Stanford HIVDB genotypic sensitivity scores yielded similar ORs for virological failure, sensitivities, specificities, and diagnostic ORs.INTERPRETATION: Ultrasensitive resistance testing for pretreatment drug resistance improved identification of people at risk of virological failure; however, this came with a reduction in our ability to identify people with viral suppression, especially at very low thresholds. Further modelling is needed to estimate the optimal trade-off for the 5% and 20% thresholds, balancing improved case finding against unnecessary regimen switching.FUNDING: The Netherlands Ministry of Foreign Affairs, IrsiCaixa, and European Union.

Objectives: The objective of this study was to define the natural genotypic variation of the HIV-1 integrase gene across Europe for epidemiological surveillance of integrase strand-transfer inhibitor (InSTI) resistance. Methods: This was a multicentre, cross-sectional study within the European SPREAD HIV resistance surveillance programme. A representative set of 300 samples was selected from 1950 naive HIV-positive subjects newly diagnosed in 2006-07. The prevalence of InSTI resistance was evaluated using quality-controlled baseline population sequencing of integrase. Signature raltegravir, elvitegravir and dolutegravir resistance mutations were defined according to the IAS-USA 2014 list. In addition, all integrase substitutions relative to HXB2 were identified, including those with a Stanford HIVdb score >= 10 to at least one InSTI. To rule out circulation of minority InSTI-resistant HIV, 65 samples were selected for 454 integrase sequencing. Results: For the population sequencing analysis, 278 samples were retrieved and successfully analysed. No signature resistance mutations to any of the InSTIs were detected. Eleven (4%) subjects had mutations at resistance-associated positions with an HIVdb score >= 10. Of the 56 samples successfully analysed with 454 sequencing, no InSTI signature mutations were detected, whereas integrase substitutions with an HIVdb score >= 10 were found in 8 (14.3%) individuals. Conclusions: No signature InSTI-resistant variants were circulating in Europe before the introduction of InSTIs. However, polymorphisms contributing to InSTI resistance were not rare. As InSTI use becomes more widespread, continuous surveillance of primary InSTI resistance is warranted. These data will be key to modelling the kinetics of InSTI resistance transmission in Europe in the coming years.

Background The S282T mutation is the main variant described associated with resistance to nucleos(t)ide analogues hepatitis C virus (HCV) NS5B polymerase inhibitors. Objective We aimed here to investigate whether this substitution pre-existed in treatment naive HCV/HIV-1 coinfected patients. Study design NS5B polymerase deep sequencing was performed at a median coverage per base of 4471 in 16 patient samples. Results No S282T variant was detected in the 16 analyzed samples. Conclusion This finding is in agreement with the high genetic barrier of nucleoside analogues NS5B polymerase inhibitors and the clinical efficacy of these compounds.

Homozygosity for a 32 bp deletion in CCR5 (CCR5-Δ32/Δ32) is associated with strong resistance against HIV-1 infection. Several HLA types have been associated to improved viral control and/or delayed progression to AIDS. We report a unique HIV-1 infected individual homozygous for CCR5-Δ32/Δ32 and carrier of HLA-A*2402 and HLA-B*5701. In comparison with earlier data and although a replication competent virus has been isolated, the patient presents better immune status, response to treatment and disease evolution, which may be related to the control exerted by HLA class I restricted T cell immunity. Importantly, the accumulation of protective factors does not warrant a complete protection to HIV infection and the subsequent life-long treatment.

The emerging HIV-1 resistance epidemic is threatening the impressive global advances in HIV-1 infection treatment and prevention achieved in the last decade. Next-generation sequencing is improving our ability to understand, diagnose and prevent HIV-1 resistance, being increasingly cost-effective and more accessible. However, NGS still faces a number of limitations that need to be addressed to enable its widespread use. Here, we will review the main NGS platforms available for HIV-1 diagnosis, the factors affecting the clinical utility of NGS testing and the evidence supporting -or not- ultrasensitive genotyping over Sanger sequencing for routine HIV-1 diagnosis. Now that global HIV-1 eradication might be within our reach, making NGS accessible also to LMICs has become a priority. Reductions in sequencing costs, particularly in library preparation, and accessibility to low-cost, robust but simplified automated bioinformatic analyses of NGS data will remain essential to end the HIV-1 pandemic.

In a South African cohort of participants living with HIV developing virological failure on first-line tenofovir disoproxyl fumarate (TDF)-based regimens, at least 70% of participants demonstrated TDF resistance according to combined Sanger and MiSeq genotyping. Sanger sequencing missed the K65R mutation in 30% of samples. Unless HIV genotyping is available to closely monitor epidemiological HIV resistance to TDF, its efficacy as second-line therapy will be greatly compromised.

Objective: This article aims to investigate if the detection of preexisting drug-resistant minority variant (DRMV) and/or X4 HIV-1 variants could improve the efficacy of first-line combined antiretroviral therapy (ART) in late presenters. Design: Post-hoc, combined analysis of two open-label, prospective, randomized clinical trials comparing first-line ART with efavirenz (EFV) vs. ritonavir-boosted protease inhibitor (PI/r)-based regimens in ART-naive, HIV-1-infected patients, with CD4(+) T-cell counts less than 100 cells/mu l and wild-type HIV-1 by bulk sequencing. Methods: Pre-ART samples were reanalyzed for the presence of DRMVs and X4 HIV-1 using 454 sequencing. Kaplan-Meier curves and Cox regression were used to evaluate the association between X4 HIV and DRMVs and risk of virological failure. Results: From 141 evaluable patients, 57 received EFV, and 84 received PI/r, including first-line ART. Median pre-ART CD4(+) T-cell counts and HIV-1 RNA levels were 39 cells/mu l and 257-424 copies/ml, respectively; 35.5% of patients had X4 HIV variants. Detection of DRMVs leading to an ART-specific cumulative HIVdb score of at least 10 increased the risk of virological failure in patients initiating EFV [log-rank P = 0.048, hazard ratio = 4.3 (95% confidence interval: 0.8, 25.0), P = 0.074], but not in those starting PI/r. Presence of X4 HIV did not affect virological outcomes, but was associated with impaired CD4(+) T-cell count recovery over 2 years (214 vs. 315 cells/mu l with X4 vs. R5 HIV-1 tropism, respectively, P = 0.017). Conclusion: Accounting for preexisting DRMVs may improve the outcomes of first-line nonnucleoside reverse transcriptase inhibitor-based ART in late presenters with advanced immune suppression. Presence of X4 HIV-1 at diagnosis predicts impaired immune restoration under ART. Copyright (C) 2015 Wolters Kluwer Health, Inc. All rights reserved.

Background. The clinical relevance of ultrasensitive human immunodeficiency virus type 1 (HIV-1) genotypicresistance testing in antiretroviral treatment (ART)-experienced individuals remains unknown.Methods. Thiswasa retrospective,multicentre,cohortstudy inART-experienced, HIV-1-infectedadultswho ini-tiated salvage ART including, at least 1 ritonavir-boosted protease inhibitor, raltegravir or etravirine. Presalvage ARTSangerand454sequencingofplasmaHIV-1wereusedtogenerateseparategenotypicsensitivityscores(GSS)usingtheHIVdb, ANRS, and REGA algorithms. Virological failure (VF) was defined as 2 consecutive HIV-1 RNA levels ≥200copies/mL at least 12 weeks after salvage ART initiation, whereas subjects remained on the same ART. The ability ofSangerand454-GSStopredictVFwasassessedbyreceiveroperatingcharacteristic(ROC)curvesandsurvivalanalyses.Results. Thestudyincluded132evaluablesubjects;28(21%)developedVF.UsingHIVdb,454predictedVFbetterthanSangersequencingintheROCcurveanalysis(areaunderthecurve:0.69vs0.60,DelongtestP=.029).TimetoVFwas shorter for subjects with 454-GSS

BACKGROUND: A sensitive, phenotypic reverse transcriptase (RT)-based drug susceptibility assay for the detection of etravirine (ETR) resistance in patient isolates was developed and compared with the results from direct sequencing and ultra-deep pyrosequencing (UDPS). METHODS: Samples were obtained from 15 patients with antiretroviral therapy (ART) failure and from five non-nucleoside reverse transcriptase inhibitor (NNRTI)-naïve patients of whom four were infected by an NNRTI-resistant strain (transmitted drug resistance, TDR). In five patients, two consecutive samples (a and b) were taken for follow up of the virological response. HIV-1 RT was purified and drug susceptibility (IC50) to ETR was estimated. Direct sequencing was performed in all samples and UDPS in samples from nine patients. RESULTS: Increased IC50 to ETR was found in samples from 13 patients where direct sequencing predicted resistance in only four. UDPS identified additional (N = 11) NNRTI resistance associated mutations (RAMs) in six of nine tested patients. During early failure, IC50 increases were observed in three of six patients without any ETR-RAMs detected by direct sequencing. In further two patients, who stopped NNRTI before sampling, increased IC50 values were found shortly after, despite absence of ETR-RAMs. In two patients who had stopped NNRTI for >1 year, a concordance between phenotype and genotypes was found. Two patients with TDR had increased IC50 despite no ETR-RAMs were detected by direct sequencing. UDPS revealed additional ETR-RAMs in four patients with a discrepancy between phenotype and direct sequencing. CONCLUSIONS: The RT-based phenotypic assay showed decreased ETR susceptibility in patients where direct sequencing predicted ETR-sensitive virus. This increased phenotypic sensitivity was to a large extent supported by UDPS and treatment history. Our method could be valuable for further studies on the phenotypic kinetics of NNRTI resistance. The clinical relevance remains to be studied in larger patient-populations.

Standardization of sequence chromatogram analysis is required for consistent genotypic tropism determination across laboratories. A freely available, fast, and automated chromatogram analysis tool (RECall) provided tropism interpretations equivalent to those of manual sequence editing of 521 V3 loop HIV-1 population sequences, suggesting that RECall can be useful in standardizing genotypic tropism testing across laboratories.

OBJECTIVES To evaluate the safety and efficacy of switching the third drug of antiretroviral treatment to maraviroc in aviraemic subjects infected with R5 HIV. PATIENTS AND METHODS This is a pilot, prospective, randomized clinical trial (ClinicalTrials ID: NCT00966329). Eighty HIV-1-infected aviraemic adults on stable antiretroviral treatment for ≥1 year and no antiretroviral drug resistance were screened for the presence of non-R5 HIV by triplicate proviral V3 population sequencing. From them, 30 subjects with R5 HIV-1 were randomized 1 : 1 to switch the non-nucleoside reverse transcriptase inhibitor or ritonavir-boosted protease inhibitor to maraviroc (n = 15) or to continue the same antiretroviral treatment (controls, n = 15). The principal endpoint was the proportion of subjects with HIV-1 RNA

Introduction It is uncertain if plasma HIV-1 tropism is an independent predictor of short-term risk of clinical progression / death, in addition to the CD4 count and HIV RNA level. We conducted a nested case-control study within EuroSIDA to assess this question amongst people with current HIV RNA level >1000 copies/mL, including both people on ART and those ART naïve. Methods People with an AIDS diagnosis or who died from any causes for whom there was a stored plasma sample with HIV-1 RNA (VL)≥1,000 copies/mL available in the time window of 3–12 months prior to the event were identified. At least one control was selected for each case matched for age, VL and HCV status at the time of sampling. Controls were event-free after a matched duration of time from the date of sampling. Plasma HIV tropism was estimated using 454 and population sequencing (PS). Non-R5 HIV was defined as: (a) ≥2% of sequences with a Geno2Pheno (G2P) FPR≤3.75% by 454, and (b) a G2P FPR≤10% by PS. We also compared CD4 slopes over the 12 months following the date of sampling using a linear mixed model with random intercept according to HIV tropism and ART status. Results The study included 266 subjects, 100 cases and 166 controls, with sample taken on average in 2006; 23% and 24% had non-R5 HIV by 454 and PS respectively. There were 19% women, 25% MSM, 92% Caucasians, 22% HCV+. At the time of sampling, 26% were ART-naïve, 25% had started but were off ART and 49% were receiving ART. The median age, CD4 and viral load was 41 years, 350 cells/mm3 and 4.81 log c/mL, respectively. Baseline characteristics were well balanced by tropism. Factors independently associated with clinical progression or death were female gender (OR=2.12; 95% CI=1.04, 4.36; p=0.038), CD4+ count (OR=0.90 per 100 cells/mm3 higher; 95% CI 0.80, 1.00; p=0.058), being on ART (OR=2.72; 95% CI 1.15, 6.41; p=0.022) and calendar year of sample (OR=0.84 per more recent year; 95% CI=0.77, 0.91; p

Background There is legitimate concern that minority drug-resistant mutants may be selected during the initial HIV-1 RNA decay phase following antiretroviral therapy initiation, thus undermining efficacy of treatment. The goal of this study was to characterize viral resistance emergence and address viral population evolution during the first phase of viral decay after treatment containing initiation. Findings 454 sequencing was used to characterize viral genetic diversity and polymorphism composition of the HIV-1 integrase gene during the first two weeks following initiation of raltegravir-containing HAART in four ART-experienced subjects. No low-prevalence Raltegravir (RAL) drug resistance mutations (DRM) were found at baseline. All patients undergoing treatment received a fully active ART according to GSS values (GSS ≥ 3.5). No emergence of DRM after treatment initiation was detected. Longitudinal analysis showed no evidence of any other polymorphic mutation emergence or variation in viral diversity indexes. Conclusions This suggests that fully active salvage antiretroviral therapy including raltegravir achieves a complete blockade of HIV-1 replication in plasma. It is unlikely that raltegravir-resistant HIV-1 may be selected in plasma during the early HIV-1 RNA decay after treatment initiation if the administered therapy is active enough.

Bartonella bacilliformis is the causative agent of Carrion's disease, a highly endemic human bartonellosis in Peru. We performed a whole-genome assembly of two B. bacilliformis strains isolated from the blood of infected patients in the acute phase of Carrion's disease from the Cusco and Piura regions in Peru.

Introduction : It is uncertain if plasma HIV-1 tropism is an independent predictor of short-term risk of clinical progression / death, in addition to the CD4 count and HIV RNA level. We conducted a nested case-control study within EuroSIDA to assess this question amongst people with current HIV RNA level >1000 copies/mL, including both people on ART and those ART naive. Methods : People with an AIDS diagnosis or who died from any causes for whom there was a stored plasma sample with HIV-1 RNA (VL)≥1,000 copies/mL available in the time window of 3–12 months prior to the event were identified. At least one control was selected for each case matched for age, VL and HCV status at the time of sampling. Controls were event-free after a matched duration of time from the date of sampling. Plasma HIV tropism was estimated using 454 and population sequencing (PS). Non-R5 HIV was defined as: (a) ≥2% of sequences with a Geno2Pheno (G2P) FPR≤3.75% by 454, and (b) a G2P FPR≤10% by PS. We also compared CD4 slopes over the 12 months following the date of sampling using a linear mixed model with random intercept according to HIV tropism and ART status. Results : The study included 266 subjects, 100 cases and 166 controls, with sample taken on average in 2006; 23% and 24% had non-R5 HIV by 454 and PS respectively. There were 19% women, 25% MSM, 92% Caucasians, 22% HCV+. At the time of sampling, 26% were ART-naive, 25% had started but were off ART and 49% were receiving ART. The median age, CD4 and viral load was 41 years, 350 cells/mm 3 and 4.81 log c/mL, respectively. Baseline characteristics were well balanced by tropism. Factors independently associated with clinical progression or death were female gender (OR=2.12; 95% CI=1.04, 4.36; p=0.038), CD4+ count (OR=0.90 per 100 cells/mm 3 higher; 95% CI 0.80, 1.00; p=0.058), being on ART (OR=2.72; 95% CI 1.15, 6.41; p=0.022) and calendar year of sample (OR=0.84 per more recent year; 95% CI=0.77, 0.91; p