Significant Impact of Coronavirus Disease 2019 (COVID-19) on Human Immunodeficiency Virus (HIV) Care in Hospitals Affecting the First Pillar of the HIV Care Continuum.

During COVID-19 lockdown, the in-hospital number of HIV indicator conditions decreased disproportionally compared with other non-COVID-19 diseases, which was accompanied by reduced HIV testing rates, number and proportion of positive HIV tests, and new HIV referrals, with more late presentation after lockdown cessation, indicating a significantly impacted HIV care continuum.

Viral Kinetics and Resistance Development in Children Treated with Neuraminidase Inhibitors: The Influenza Resistance Information Study (IRIS).

BACKGROUND: We studied the effect of age, baseline viral load, vaccination status, antiviral therapy, and emergence of drug resistance on viral shedding in children infected with influenza A or B virus. METHODS: Samples from children (aged ≤13 years) enrolled during the 7 years of the prospective Influenza Resistance Information Study were analyzed using polymerase chain reaction to determine the influenza virus (sub-)type, viral load, and resistance mutations. Disease severity was assessed; clinical symptoms were recorded. The association of age with viral load and viral clearance was examined by determining the area under the curve for viral RNA shedding using logistic regression and Kaplan-Meier analyses. RESULTS: A total of 2131 children infected with influenza (683, A/H1N1pdm09; 825, A/H3N2; 623, influenza B) were investigated. Age did not affect the mean baseline viral load. Children aged 1-5 years had prolonged viral RNA shedding (±1-2 days) compared with older children and up to 1.2-fold higher total viral burden. Besides, in older age (odds ratio [OR], 1.08; confidence interval [CI], 1.05-1.12), prior vaccination status (OR, 1.72; CI, 1.22-2.43) and antiviral treatment (OR, 1.74; CI, 1.43-2.12) increased the rate of viral clearance. Resistance mutations were detected in 49 children infected with influenza A virus (34, A/H1N1pdm09; 15, A/H3N2) treated with oseltamivir, most of whom were aged <5 years (n = 39). CONCLUSIONS: Children aged 1-5 years had a higher total viral burden with prolonged virus shedding and had an increased risk of acquiring resistance mutations following antiviral treatment. CLINICAL TRIALS REGISTRATION: NCT00884117.

Transmission of NS5A-Inhibitor Resistance-Associated Substitutions Among Men Who Have Sex With Men Recently Infected with Hepatitis C Virus Genotype 1a.

The transmission of direct-acting antiviral resistance-associated substitutions (RAS) could hamper hepatitis C virus (HCV) cure rates and elimination efforts. A phylogenetic analysis of 87 men who have sex with men recently infected with HCV genotype 1a placed one-third (28/87) in a large cluster, in which 96% harbored NS5A M28V RAS.

Cost-effectiveness and budget effect of pre-exposure prophylaxis for HIV-1 prevention in Germany from 2018 to 2058.

BackgroundPre-exposure prophylaxis (PrEP) is a highly effective HIV prevention strategy for men-who-have-sex-with-men (MSM). The high cost of PrEP has until recently been a primary barrier to its use. In 2017, generic PrEP became available, reducing the costs by 90%.AimOur objective was to assess cost-effectiveness and costs of introducing PrEP in Germany.MethodsWe calibrated a deterministic mathematical model to the human immunodeficiency virus (HIV) epidemic among MSM in Germany. PrEP was targeted to 30% of high-risk MSM. It was assumed that PrEP reduces the risk of HIV infection by 85%. Costs were calculated from a healthcare payer perspective using a 40-year time horizon starting in 2018.ResultsPrEP can avert 21,000 infections (interquartile range (IQR): 16,000-27,000) in the short run (after 2 years scale-up and 10 years full implementation). HIV care is predicted to cost EUR 36.2 billion (IQR: 32.4-40.4 billion) over the coming 40 years. PrEP can increase costs by at most EUR 150 million within the first decade after introduction. Ten years after introduction, PrEP can become cost-saving, accumulating to savings of HIV-related costs of EUR 5.1 billion (IQR: 3.5-6.9 billion) after 40 years. In a sensitivity analysis, PrEP remained cost-saving even at a 70% price reduction of antiretroviral drug treatment and a lower effectiveness of PrEP.ConclusionIntroduction of PrEP in Germany is predicted to result in substantial health benefits because of reductions in HIV infections. Short-term financial investments in providing PrEP will result in substantial cost-savings in the long term.

HIV-1 Resistance Dynamics in Patients With Virologic Failure to Dolutegravir Maintenance Monotherapy.

Background: A high genetic barrier to resistance to the integrase strand transfer inhibitor (INSTI) dolutegravir has been reported in vitro and in vivo. We describe the dynamics of INSTI resistance-associated mutations (INSTI-RAMs) and mutations in the 3'-polypurine tract (3'-PPT) in relation to virologic failure (VF) observed in the randomized Dolutegravir as Maintenance Monotherapy for HIV-1 study (DOMONO, NCT02401828). Methods: From 10 patients with VF, plasma samples were collected before the start of cART and during VF, and were used to generate Sanger sequences of integrase, the 5' terminal bases of the 3' long terminal repeat (LTR), and the 3'-PPT. Results: Median human immunodeficiency virus RNA load at VF was 3490 copies/mL (interquartile range 1440-4990 copies/mL). INSTI-RAMs (S230R, R263K, N155H, and E92Q+N155H) were detected in 4 patients, no INSTI-RAMs were detected in 4 patients, and sequencing of the integrase gene was unsuccessful in 2 patients. The time to VF ranged from 4 weeks to 72 weeks. In 1 patient, mutations developed in the highly conserved 3'-PPT. No changes in the terminal bases of the 3'-LTR were observed. Conclusions: The genetic barrier to resistance is too low to justify dolutegravir maintenance monotherapy because single INSTI-RAMs are sufficient to cause VF. The large variation in time to VF suggests that stochastic reactivation of a preexisting provirus containing a single INSTI-RAM is the mechanism for failure. Changes in the 3'-PPT point to a new dolutegravir resistance mechanism in vivo. Clinical Trials Registration: NCT02401828.

The S230R Integrase Substitution Associated With Virus Load Rebound During Dolutegravir Monotherapy Confers Low-Level Resistance to Integrase Strand-Transfer Inhibitors.

Background: Dolutegravir (DTG) is an integrase strand-transfer inhibitor (INSTI) used for treatment of human immunodeficiency virus (HIV)-infected individuals. Owing to its high genetic barrier to resistance, DTG has been clinically investigated as maintenance monotherapy to maintain viral suppression and to reduce complication and healthcare costs. Our study aims to explain the underlying mechanism related to the emergence of a S230R substitution in patients who experienced virologic failure while using DTG monotherapy. Methods: We evaluated the effect of the S230R substitution in regard to integrase enzyme activity, viral infectivity, replicative capacity, and susceptibility to different INSTIs by biochemical and cell-based assays. Results: The S230R substitution conferred a 63% reduction in enzyme efficiency. S230R virus was 1.29-fold less infectious than wild-type virus but could replicate in PM1 cells without significant delay. Resistance levels against DTG, cabotegravir, raltegravir, and elvitegravir in tissue culture were 3.85-, 3.72-, 1.52-, and 1.21-fold, respectively, in virus with the S230R substitution. Conclusions: Our data indicate that the S230R substitution is comparable to the previously reported R263K substitution in some respects. Virologic failure during DTG monotherapy can occur through the development of the S230R or R263K mutation, without the need for high-level DTG resistance.

Declining Hepatitis C Virus (HCV) Incidence in Dutch Human Immunodeficiency Virus-Positive Men Who Have Sex With Men After Unrestricted Access to HCV Therapy.

Background: Direct-acting antivirals (DAAa) cure hepatitis C virus (HCV) infections in 95% of infected patients. Modeling studies predict that universal HCV treatment will lead to a decrease in the incidence of new infections but real-life data are lacking. The incidence of HCV among Dutch human immunodeficiency virus (HIV)-positive men who have sex with men (MSM) has been high for >10 years. In 2015 DAAs became available to all Dutch HCV patients and resulted in a rapid treatment uptake in HIV-positive MSM. We assessed whether this uptake was followed by a decrease in the incidence of HCV infections. Methods: Two prospective studies of treatment for acute HCV infection enrolled patients in 17 Dutch HIV centers, having 76% of the total HIV-positive MSM population in care in the Netherlands. Patients were recruited in 2014 and 2016, the years before and after unrestricted DAA availability. We compared the HCV incidence in both years. Results: The incidence of acute HCV infection decreased from 93 infections during 8290 person-years of follow-up (PYFU) in 2014 (11.2/1000 PYFU; 95% confidence interval [CI], 9.1-13.7) to 49 during 8961 PYFU in 2016 (5.5/1000 PYFU; 4.1-7.2). The incidence rate ratio of 2016 compared with 2014 was 0.49 (95% CI, .35-.69). Simultaneously, a significant increase in the percentage positive syphilis (+2.2%) and gonorrhea (+2.8%) tests in HIV-positive MSM was observed at sexual health clinics across the Netherlands and contradicts a decrease in risk behavior as an alternative explanation. Conclusions: Unrestricted DAA availability in the Netherlands was followed by a 51% decrease in acute HCV infections among HIV-positive MSM.

Immune-escape mutations and stop-codons in HBsAg develop in a large proportion of patients with chronic HBV infection exposed to anti-HBV drugs in Europe.

BACKGROUND: HBsAg 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. METHODS: This 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. RESULTS: At 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. CONCLUSIONS: Immune-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.

Outcomes and Susceptibility to Neuraminidase Inhibitors in Individuals Infected With Different Influenza B Lineages: The Influenza Resistance Information Study.

BACKGROUND: Little is known about how influenza infections caused by B/Victoria and B/Yamagata virus lineages compare with respect to disease course and susceptibility to antiviral therapy. METHODS: Data from patients with influenza B infections from the first 5 years (2009-2013) of the prospective Influenza Resistance Information Study (IRIS, NCT00884117) were evaluated. Cultured viruses were phenotypically tested for neuraminidase inhibitor (NAI) sensitivity, and sequenced to determine virus lineage (B/Victoria or B/Yamagata). Differences in clinical outcomes (viral clearance and symptom resolution) between virus lineages were assessed using Kaplan-Meier analysis. RESULTS: In all, 914 patients were positive for influenza B by reverse transcriptase polymerase chain reaction ( RT-PCR: B/Victoria, 586; B/Yamagata, 289; not subtyped, 39); 474 were treated with antivirals. No phenotypic resistance to oseltamivir or zanamivir was found in B/Victoria or B/Yamagata viruses. Of 15 predefined resistance mutations, 2 were detected by neuraminidase sequencing: I221T had reduced sensitivity to oseltamivir, and I221V was sensitive to NAI inhibition. No consistent differences between virus lineages in times to viral clearance or to symptom or fever resolution were found in adults and adolescents or in children. CONCLUSIONS: Influenza B virus lineage had no notable effect on disease outcomes or antiviral susceptibility in this population.

Combined Analysis of the Prevalence of Drug-Resistant Hepatitis B Virus in Antiviral Therapy-Experienced Patients in Europe (CAPRE).

BACKGROUND: European guidelines recommend treatment of chronic hepatitis B virus infection (CHB) with the nucleos(t)ide analogs (NAs) entecavir or tenofovir. However, many European CHB patients have been exposed to other NAs, which are associated with therapy failure and resistance. The CAPRE study was performed to gain insight in prevalence and characteristics of NA resistance in Europe. METHODS: A survey was performed on genotypic resistance testing results acquired during routine monitoring of CHB patients with detectable serum hepatitis B virus DNA in European tertiary referral centers. RESULTS: Data from 1568 patients were included. The majority (73.8%) were exposed to lamivudine monotherapy. Drug-resistant strains were detected in 52.7%. The most frequently encountered primary mutation was M204V/I (48.7%), followed by A181T/V (3.8%) and N236T (2.6%). In patients exposed to entecavir (n = 102), full resistance was present in 35.3%. Independent risk factors for resistance were age, viral load, and lamivudine exposure (P < .001). CONCLUSIONS: These findings support resistance testing in cases of apparent NA therapy failure. This survey highlights the impact of exposure to lamivudine and adefovir on development of drug resistance and cross-resistance. Continued use of these NAs needs to be reconsidered at a pan-European level.

Transmission of HIV Drug Resistance and the Predicted Effect on Current First-line Regimens in Europe.

BACKGROUND: Numerous studies have shown that baseline drug resistance patterns may influence the outcome of antiretroviral therapy. Therefore, guidelines recommend drug resistance testing to guide the choice of initial regimen. In addition to optimizing individual patient management, these baseline resistance data enable transmitted drug resistance (TDR) to be surveyed for public health purposes. The SPREAD program systematically collects data to gain insight into TDR occurring in Europe since 2001. METHODS: Demographic, clinical, and virological data from 4140 antiretroviral-naive human immunodeficiency virus (HIV)-infected individuals from 26 countries who were newly diagnosed between 2008 and 2010 were analyzed. Evidence of TDR was defined using the WHO list for surveillance of drug resistance mutations. Prevalence of TDR was assessed over time by comparing the results to SPREAD data from 2002 to 2007. Baseline susceptibility to antiretroviral drugs was predicted using the Stanford HIVdb program version 7.0. RESULTS: The overall prevalence of TDR did not change significantly over time and was 8.3% (95% confidence interval, 7.2%-9.5%) in 2008-2010. The most frequent indicators of TDR were nucleoside reverse transcriptase inhibitor (NRTI) mutations (4.5%), followed by nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations (2.9%) and protease inhibitor mutations (2.0%). Baseline mutations were most predictive of reduced susceptibility to initial NNRTI-based regimens: 4.5% and 6.5% of patient isolates were predicted to have resistance to regimens containing efavirenz or rilpivirine, respectively, independent of current NRTI backbones. CONCLUSIONS: Although TDR was highest for NRTIs, the impact of baseline drug resistance patterns on susceptibility was largest for NNRTIs. The prevalence of TDR assessed by epidemiological surveys does not clearly indicate to what degree susceptibility to different drug classes is affected.

Dolutegravir as maintenance monotherapy: first experiences in HIV-1 patients.

BACKGROUND: Dolutegravir is recommended as part of combination ART (cART) for HIV-1-infected patients. Toxicities, drug interactions and costs related to cART still warrant the search for improved treatment options. Dolutegravir's high resistance barrier might make it suitable as antiretroviral maintenance monotherapy. The feasibility of this strategy is currently unknown. METHODS: This is a prospective case series on five consecutive HIV-1-infected patients on cART without previous virological failure who switched to dolutegravir monotherapy. All were HIV-RNA suppressed <50 copies/mL and had contraindications to current and alternative combinations of antiretroviral drugs. HIV-RNA was measured at baseline, week 4, week 8, week 12 and every 6 weeks thereafter. Patients would be switched back to their original cART upon confirmed HIV-RNA >50 copies/mL. RESULTS: The five patients had been HIV-RNA suppressed <50 copies/mL for ≥1.5 years prior to the initiation of dolutegravir monotherapy. All were on NNRTI-containing regimens at the switch. HIV-RNA remained <50 copies/mL at all timepoints in four patients. One patient, with end-stage renal disease and on calcium supplements, had a pre-cART HIV-RNA of 625 000 copies/mL with a CD4 nadir of 120 cells/mm(3) and had HIV-RNA of 8150 copies/mL at week 30. The dolutegravir Ctrough was 0.18 mg/L. This patient did not have acquired resistance or evidence of adherence problems and HIV-RNA was resuppressed after switching to his former cART. CONCLUSIONS: This case series indicates that dolutegravir monotherapy might be a valuable maintenance option in selected HIV-infected patients who are well suppressed on cART, if confirmed by future randomized clinical trials.

Mutation V111I in HIV-2 reverse transcriptase increases the fitness of the nucleoside analogue-resistant K65R and Q151M viruses.

UNLABELLED: Infection with HIV-2 can ultimately lead to AIDS, although disease progression is much slower than with HIV-1. HIV-2 patients are mostly treated with a combination of nucleoside reverse transcriptase (RT) inhibitors (NRTIs) and protease inhibitors designed for HIV-1. Many studies have described the development of HIV-1 resistance to NRTIs and identified mutations in the polymerase domain of RT. Recent studies have shown that mutations in the connection and RNase H domains of HIV-1 RT may also contribute to resistance. However, only limited information exists regarding the resistance of HIV-2 to NRTIs. In this study, therefore, we analyzed the polymerase, connection, and RNase H domains of RT in HIV-2 patients failing NRTI-containing therapies. Besides the key resistance mutations K65R, Q151M, and M184V, we identified a novel mutation, V111I, in the polymerase domain. This mutation was significantly associated with mutations K65R and Q151M. Sequencing of the connection and RNase H domains of the HIV-2 patients did not reveal any of the mutations that were reported to contribute to NRTI resistance in HIV-1. We show that V111I does not strongly affect drug susceptibility but increases the replication capacity of the K65R and Q151M viruses. Biochemical assays demonstrate that V111I restores the polymerization defects of the K65R and Q151M viruses but negatively affects the fidelity of the HIV-2 RT enzyme. Molecular dynamics simulations were performed to analyze the structural changes mediated by V111I. This showed that V111I changed the flexibility of the 110-to-115 loop region, which may affect deoxynucleoside triphosphate (dNTP) binding and polymerase activity. IMPORTANCE: Mutation V111I in the HIV-2 reverse transcriptase enzyme was identified in patients failing therapies containing nucleoside analogues. We show that the V111I change does not strongly affect the sensitivity of HIV-2 to nucleoside analogues but increases the fitness of viruses with drug resistance mutations K65R and Q151M.

Global Dispersal Pattern of HIV Type 1 Subtype CRF01_AE: A Genetic Trace of Human Mobility Related to Heterosexual Sexual Activities Centralized in Southeast Asia.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) subtype CRF01_AE originated in Africa and then passed to Thailand, where it established a major epidemic. Despite the global presence of CRF01_AE, little is known about its subsequent dispersal pattern. METHODS: We assembled a global data set of 2736 CRF01_AE sequences by pooling sequences from public databases and patient-cohort studies. We estimated viral dispersal patterns, using statistical phylogeographic analysis run over bootstrap trees estimated by the maximum likelihood method. RESULTS: We show that Thailand has been the source of viral dispersal to most areas worldwide, including 17 of 20 sampled countries in Europe. Japan, Singapore, Vietnam, and other Asian countries have played a secondary role in the viral dissemination. In contrast, China and Taiwan have mainly imported strains from neighboring Asian countries, North America, and Africa without any significant viral exportation. DISCUSSION: The central role of Thailand in the global spread of CRF01_AE can be probably explained by the popularity of Thailand as a vacation destination characterized by sex tourism and by Thai emigration to the Western world. Our study highlights the unique case of CRF01_AE, the only globally distributed non-B clade whose global dispersal did not originate in Africa.

Geographic and temporal trends in the molecular epidemiology and genetic mechanisms of transmitted HIV-1 drug resistance: an individual-patient- and sequence-level meta-analysis.

BACKGROUND: Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes. METHODS AND FINDINGS: We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naïve individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05-1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06-1.25), North America (OR = 1.19; 95% CI: 1.12-1.26), Europe (OR = 1.07; 95% CI: 1.01-1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12-1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92-1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions­a proxy for recent infection­yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs­K101E, K103N, Y181C, and G190A­accounted for >80% of NNRTI-associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI-associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high-level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling. CONCLUSIONS: Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen.

Infection with the frequently transmitted HIV-1 M41L variant has no influence on selection of tenofovir resistance.

OBJECTIVES: In ∼10% of newly diagnosed HIV-1 patients, drug-resistant viral variants are detected. In such transmitted HIV-1 variants, the thymidine analogue mutation (TAM) M41L is frequently observed as a single resistance mutation and these viral variants often belong to phylogenetic transmission clusters. The presence of at least three TAMs, in particular patterns with M41L/L210W, impairs the efficacy of the extensively used drug tenofovir. We investigated whether the presence of a single M41L mutation at baseline influences the selection of resistance to tenofovir and emtricitabine in vitro and in vivo. METHODS: The impact of M41L on the development of drug resistance to tenofovir and emtricitabine was determined by extensive in vitro selection experiments and investigation of the virological outcome of patients on a first-line regimen. RESULTS: The presence of a single M41L mutation did not influence the selected mutational profile or the genetic barrier to resistance to tenofovir and/or emtricitabine during long-term in vitro selection experiments. In vivo, virological outcome of first-line regimens containing tenofovir and emtricitabine was comparable between patients diagnosed with HIV-1 harbouring M41L (n=17, 16 were part of one transmission cluster) and WT virus (n=248). CONCLUSIONS: Detection of a single M41L reverse transcriptase mutation at baseline did not influence the development of resistance in vitro or virological outcome on tenofovir-containing regimens in patients belonging to a large transmission cluster. Our results indicate that a high genetic barrier regimen may not be required when patients are diagnosed with HIV variants containing a single M41L mutation in reverse transcriptase.

Diminished transmission of drug resistant HIV-1 variants with reduced replication capacity in a human transmission model.

BACKGROUND: Different patterns of drug resistance are observed in treated and therapy naïve HIV-1 infected populations. Especially the NRTI-related M184I/V variants, which are among the most frequently encountered mutations in treated patients, are underrepresented in the antiretroviral naïve population. M184I/V mutations are known to have a profound effect on viral replication and tend to revert over time in the new host. However it is debated whether a diminished transmission efficacy of HIV variants with a reduced replication capacity can also contribute to the observed discrepancy in genotypic patterns. As dendritic cells (DCs) play a pivotal role in HIV-1 transmission, we used a model containing primary human Langerhans cells (LCs) and DCs to compare the transmission efficacy M184 variants (HIV-M184V/I/T) to HIV wild type (HIV-WT). As control, we used HIV harboring the NNRTI mutation K103N (HIV-K103N) which has a minor effect on replication and is found at a similar prevalence in treated and untreated individuals. RESULTS: In comparison to HIV-WT, the HIV-M184 variants were less efficiently transmitted to CCR5(+) Jurkat T cells by both LCs and DCs. The transmission rate of HIV-K103N was slightly reduced to HIV-WT in LCs and even higher than HIV-WT in DCs. Replication experiments in CCR5(+) Jurkat T cells revealed no apparent differences in replication capacity between the mutant viruses and HIV-WT. However, viral replication in LCs and DCs was in concordance with the transmission results; replication by the HIV-M184 variants was lower than replication by HIV-WT, and the level of replication of HIV-K103N was intermediate for LCs and higher than HIV-WT for DCs. CONCLUSIONS: Our data demonstrate that drug resistant M184-variants display a reduced replication capacity in LCs and DCs which directly impairs their transmission efficacy. As such, diminished transmission efficacy may contribute to the lower prevalence of drug resistant variants in therapy naive individuals.

Persistence of frequently transmitted drug-resistant HIV-1 variants can be explained by high viral replication capacity.

BACKGROUND: In approximately 10% of newly diagnosed individuals in Europe, HIV-1 variants harboring transmitted drug resistance mutations (TDRM) are detected. For some TDRM it has been shown that they revert to wild type while other mutations persist in the absence of therapy. To understand the mechanisms explaining persistence we investigated the in vivo evolution of frequently transmitted HIV-1 variants and their impact on in vitro replicative capacity. RESULTS: We selected 31 individuals infected with HIV-1 harboring frequently observed TDRM such as M41L or K103N in reverse transcriptase (RT) or M46L in protease. In all these samples, polymorphisms at non-TDRM positions were present at baseline (median protease: 5, RT: 6). Extensive analysis of viral evolution of protease and RT demonstrated that the majority of TDRM (51/55) persisted for at least a year and even up to eight years in the plasma. During follow-up only limited selection of additional polymorphisms was observed (median: 1).To investigate the impact of frequently observed TDRM on the replication capacity, mutant viruses were constructed with the most frequently encountered TDRM as site-directed mutants in the genetic background of the lab strain HXB2. In addition, viruses containing patient-derived protease or RT harboring similar TDRM were made. The replicative capacity of all viral variants was determined by infecting peripheral blood mononuclear cells and subsequently monitoring virus replication. The majority of site-directed mutations (M46I/M46L in protease and M41L, M41L + T215Y and K103N in RT) decreased viral replicative capacity; only protease mutation L90M did not hamper viral replication. Interestingly, most patient-derived viruses had a higher in vitro replicative capacity than the corresponding site-directed mutant viruses. CONCLUSIONS: We demonstrate limited in vivo evolution of protease and RT harbouring frequently observed TDRM in the plasma. This is in line with the high in vitro replication capacity of patient-derived viruses harbouring TDRM compared to site-directed mutant viruses harbouring TDRM. As site-directed mutant viruses have a lower replication capacity than the patient-derived viruses with similar mutational patterns, we propose that (baseline) polymorphisms function as compensatory mutations improving viral replication capacity.

H1N1 2009 pandemic influenza virus: resistance of the I223R neuraminidase mutant explained by kinetic and structural analysis.

Two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that antiviral resistant viruses emerge and spread in the human population. The 2009 pandemic H1N1 virus is already resistant to adamantanes. Recently, a novel neuraminidase inhibitor resistance mutation I223R was identified in the neuraminidase of this subtype. To understand the resistance mechanism of this mutation, the enzymatic properties of the I223R mutant, together with the most frequently observed resistance mutation, H275Y, and the double mutant I223R/H275Y were compared. Relative to wild type, K(M) values for MUNANA increased only 2-fold for the single I223R mutant and up to 8-fold for the double mutant. Oseltamivir inhibition constants (K(I)) increased 48-fold in the single I223R mutant and 7500-fold in the double mutant. In both cases the change was largely accounted for by an increased dissociation rate constant for oseltamivir, but the inhibition constants for zanamivir were less increased. We have used X-ray crystallography to better understand the effect of mutation I223R on drug binding. We find that there is shrinkage of a hydrophobic pocket in the active site as a result of the I223R change. Furthermore, R223 interacts with S247 which changes the rotamer it adopts and, consequently, binding of the pentoxyl substituent of oseltamivir is not as favorable as in the wild type. However, the polar glycerol substituent present in zanamivir, which mimics the natural substrate, is accommodated in the I223R mutant structure in a similar way to wild type, thus explaining the kinetic data. Our structural data also show that, in contrast to a recently reported structure, the active site of 2009 pandemic neuraminidase can adopt an open conformation.

Increase in transmitted resistance to non-nucleoside reverse transcriptase inhibitors among newly diagnosed HIV-1 infections in Europe.

BACKGROUND: One out of ten newly diagnosed patients in Europe was infected with a virus carrying a drug resistant mutation. We analysed the patterns over time for transmitted drug resistance mutations (TDRM) using data from the European Spread program. METHODS: Clinical, epidemiological and virological data from 4317 patients newly diagnosed with HIV-1 infection between 2002 and 2007 were analysed. Patients were enrolled using a pre-defined sampling strategy. RESULTS: The overall prevalence of TDRM in this period was 8.9% (95% CI: 8.1-9.8). Interestingly, significant changes over time in TDRM caused by the different drug classes were found. Whereas nucleoside resistance mutations remained constant at 5%, a significant decline in protease inhibitors resistance mutations was observed, from 3.9% in 2002 to 1.6% in 2007 (p = 0.001). In contrast, resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) doubled from 2.0% in 2002 to 4.1% in 2007 (p = 0.004) with 58% of viral strains carrying a K103N mutation. Phylogenetic analysis showed that these temporal changes could not be explained by large clusters of TDRM. CONCLUSION: During the years 2002 to 2007 transmitted resistance to NNRTI has doubled to 4% in Europe. The frequent use of NNRTI in first-line regimens and the clinical impact of NNRTI mutations warrants continued monitoring.