Efficacy of tenofovir and efavirenz in combination with lamivudine or emtricitabine in antiretroviral-naive patients in Europe.

BACKGROUND: The combination of tenofovir and efavirenz with either lamivudine or emtricitabine (TELE) has proved to be highly effective in clinical trials for first-line treatment of HIV-1 infection. However, limited data are available on its efficacy in routine clinical practice. METHODS: A multicentre cohort study was performed in therapy-naive patients initiating ART with TELE before July 2009. Efficacy was studied using ITT (missing or switch = failure) and on-treatment (OT) analyses. Genotypic susceptibility scores (GSSs) were determined using the Stanford HIVdb algorithm. RESULTS: Efficacy analysis of 1608 patients showed virological suppression to <50 copies/mL at 48 weeks in 91.5% (OT) and 70.6% (ITT). Almost a quarter of all patients (22.9%) had discontinued TELE at week 48, mainly due to CNS toxicity. Virological failure within 48 weeks was rarely observed (3.3%, n = 53). In multilevel, multivariate analysis, infection with subtype B (P = 0.011), baseline CD4 count <200 cells/mm³ (P < 0.001), GSS <3 (P = 0.002) and use of lamivudine (P < 0.001) were associated with a higher risk of virological failure. After exclusion of patients using co-formulated compounds, virological failure was still more often observed with lamivudine. Following virological failure, three-quarters of patients switched to a PI-based regimen with GSS <3. After 1 year of second-line therapy, viral load was suppressed to <50 copies/mL in 73.5% (OT). CONCLUSIONS: In clinical practice, treatment failure on TELE regimens is relatively frequent due to toxicity. Virological failure is rare and more often observed with lamivudine than with emtricitabine. Following virological failure on TELE, PI-based second-line therapy was often successful despite GSS <3.

Primary resistance to integrase strand-transfer inhibitors in Europe.

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.

Deep sequencing does not reveal additional transmitted mutations in patients diagnosed with HIV-1 variants with single nucleoside reverse transcriptase inhibitor resistance mutations.

OBJECTIVES: The aim of the study was to gain more insight into the relationship between transmitted singletons found at HIV diagnosis by population sequencing and the possible presence of clinically relevant viral minorities containing additional resistance mutations. METHODS: We studied the viral quasispecies and therapy response in 10 individuals with transmitted single nucleoside reverse transcriptase inhibitor (NRTI)-related resistance mutations as detected by population sequencing. RESULTS: Ultra-deep pyrosequencing did not reveal additional drug-resistance mutations in nine of 10 patients. In these nine patients, no breakthrough with resistant viruses was observed despite the use of low genetic nonnucleoside reverse transcriptase inhibitor (NNRTI)-based regimens in the majority of patients. CONCLUSIONS: These data suggest that viral minority variants containing additional resistance mutations may be rare in patients with transmitted NRTI singletons in the Netherlands. Larger studies are required to confirm these findings and to determine the therapeutic consequences.

Leflunomide as part of the treatment for multidrug-resistant cytomegalovirus disease after lung transplantation: case report and review of the literature.

Treatment of cytomegalovirus (CMV) disease in transplant patients is challenging and, with antiviral resistance to first-line drugs, it remains uncertain which treatment algorithm to follow. Some data suggest that leflunomide, a pyrimidine synthesis inhibitor, can be used to treat resistant CMV infections. We report a 57-year-old CMV immunoglobulin-G (IgG)-seronegative woman, who received a bilateral lung transplant (LuTx) from a CMV IgG-positive donor with CMV primary disease. The CMV strain was genotypically resistant to ganciclovir, foscarnet, and cidofovir. After starting leflunomide as add-on therapy to a multidrug anti-CMV regimen, viral load declined substantially in 2 months without adverse events. This experience is discussed against the background of existing literature on the use of leflunomide as an anti-CMV agent in LuTx recipients.

Evaluation of the antiviral response to zanamivir administered intravenously for treatment of critically ill patients with pandemic influenza A (H1N1) infection.

A retrospective nationwide study on the use of intravenous (IV) zanamivir in patients receiving intensive care who were pretreated with oseltamivir in the Netherlands was performed. In 6 of 13 patients with a sustained reduction of the viral load, the median time to start IV zanamivir was 9 days (range, 4-11 days) compared with 14 days (range, 6-21 days) in 7 patients without viral load reduction (P = .052). Viral load response did not influence mortality. We conclude that IV zanamivir as late add-on therapy has limited effectiveness. The effect of an immediate start with IV zanamivir monotherapy or in combination with other drugs need to be evaluated.

HIV testing and antiretroviral treatment strategies for prevention of HIV infection: impact on antiretroviral drug resistance.

'Test and treat' is a strategy in which widespread screening for human immunodeficiency virus (HIV) is followed by immediate antiretroviral therapy for those testing positive, thereby potentially reducing infectiousness in larger cohorts of infected patients. However, there is a concern that test and treat could lead to increased the levels of transmissible drug-resistant HIV, especially if viral load and/or drug resistance is not routinely monitored. Reviews of the existing literature show that up to now, even in the absence of laboratory tests, drug resistance has not created major problems in sub-Saharan Africa. Here, we discuss the current evidence for the effectiveness of a preventive test and treat approach and the challenges and implications for daily clinical practice and public health.

Prediction of response to antiretroviral therapy by human experts and by the EuResist data-driven expert system (the EVE study).

OBJECTIVES: The EuResist expert system is a novel data-driven online system for computing the probability of 8-week success for any given pair of HIV-1 genotype and combination antiretroviral therapy regimen plus optional patient information. The objective of this study was to compare the EuResist system vs. human experts (EVE) for the ability to predict response to treatment. METHODS: The EuResist system was compared with 10 HIV-1 drug resistance experts for the ability to predict 8-week response to 25 treatment cases derived from the EuResist database validation data set. All current and past patient data were made available to simulate clinical practice. The experts were asked to provide a qualitative and quantitative estimate of the probability of treatment success. RESULTS: There were 15 treatment successes and 10 treatment failures. In the classification task, the number of mislabelled cases was six for EuResist and 6-13 for the human experts [mean±standard deviation (SD) 9.1±1.9]. The accuracy of EuResist was higher than the average for the experts (0.76 vs. 0.64, respectively). The quantitative estimates computed by EuResist were significantly correlated (Pearson r=0.695, P<0.0001) with the mean quantitative estimates provided by the experts. However, the agreement among experts was only moderate (for the classification task, inter-rater κ=0.355; for the quantitative estimation, mean±SD coefficient of variation=55.9±22.4%). CONCLUSIONS: With this limited data set, the EuResist engine performed comparably to or better than human experts. The system warrants further investigation as a treatment-decision support tool in clinical practice.

Increased cell division but not thymic dysfunction rapidly affects the T-cell receptor excision circle content of the naive T cell population in HIV-1 infection.

Recent thymic emigrants can be identified by T cell receptor excision circles (TRECs) formed during T-cell receptor rearrangement. Decreasing numbers of TRECs have been observed with aging and in human immunodeficiency virus (HIV)-1 infected individuals, suggesting thymic impairment. Here, we show that in healthy individuals, declining thymic output will affect the TREC content only when accompanied by naive T-cell division. The rapid decline in TRECs observed during HIV-1 infection and the increase following HAART are better explained not by thymic impairment, but by changes in peripheral T-cell division rates. Our data indicate that TREC content in healthy individuals is only indirectly related to thymic output, and in HIV-1 infection is mainly affected by immune activation.

Ordered accumulation of mutations in HIV protease confers resistance to ritonavir.

Analysis of the HIV protease gene from the plasma of HIV-infected patients revealed substitutions at nine different codons selected in response to monotherapy with the protease inhibitor ritonavir. Mutants at valine-82, although insufficient to confer resistance, appeared first in most patients. Significant phenotypic resistance required multiple mutations in HIV protease, which emerged subsequently in an ordered, stepwise fashion. The appearance of resistance mutations was delayed in patients with higher plasma levels of ritonavir. Early mutants retained susceptibility to structurally diverse protease inhibitors, suggesting that dual protease inhibitor therapy might increase the duration of viral suppression.

Launching a multidisciplinary European collaboration towards a cure for HIV: The EU2Cure Consortium.

We felt the urgency to launch the EU2Cure Consortium to support research and find a cure for the human immunodeficiency virus (HIV) infection through intensified collaboration within Europe. This consortium is open to stakeholders on cure in Europe from academia and the community to connect. The aim of this consortium is to intensify the research collaboration amongst European HIV cure groups and the community and facilitate interactions with other academic and community cure consortia, private parties, and policy makers. Our main aim is to create a European research agenda, data sharing, and development of best practice for clinical and translational science to achieve breakthroughs with clinically feasible HIV cure strategies. This consortium should also enable setting up collaborative studies accessible to a broader group of people living with HIV. Besides reservoir studies, we have identified three overlapping scientific interests in the consortium that provide a starting point for further research within a European network: developing "shock and kill" cure strategies, defining HIV cure biomarkers, and connecting cure cohorts. This strategy should aid stakeholders to sustain progress in HIV cure research regardless of coincidental global health or political crises.

Antiviral resistance and impact on viral replication capacity: evolution of viruses under antiviral pressure occurs in three phases.

Resistance development is a major obstacle to antiviral therapy, and all active antiviral agents have shown to select for resistance mutations. Aspects of antiviral resistance development are discussed for specific compounds or drug classes in the previous chapters, while this chapter provides an overview regarding the evolution of different viruses (HIV, HBV, HCV, and Influenza) under pressure of antiviral therapy. Virus replication is an error prone process resulting in a large number of variants (quasispecies) in patients. Resistance evolution under suboptimal therapy can be schematically distinguished into three phases. (1) preexisting variants less sensitive to the respective drug are selected from the quasispecies population, (2) outgrowing variants acquire additional mutations increasing their resistance, and (3) compensatory mutations accumulate to overcome the generally reduced replicative capacity of resistant variants. Successful therapy should be aimed at suppression of all existing viral variants, thus preventing selection of minority species and their subsequent evolution. This implies that the amount of mutations required for first escape to the viral regimen (genetic barrier) should be larger than the expected number of mutations present in viruses in the quasispecies. Accordingly, combination therapy can achieve complete inhibition of replication for most HIV, HBV, and Influenza infected patients without resistance development. However, resistant viruses can become selected under circumstances of suboptimal antiviral therapy and these resistant viruses can be transmitted. Proper use of drugs and worldwide monitoring for the presence and spread of drug resistant viruses are therefore of utmost importance.

Oseltamivir-resistant influenza A(H1N1) viruses detected in Europe during season 2007-8 had epidemiologic and clinical characteristics similar to co-circulating susceptible A(H1N1) viruses.

During the 2007-08 influenza season, high levels of oseltamivir resistance were detected among influenza A(H1N1) viruses ina number of European countries. We used surveillance data to describe influenza A(H1N1) cases for whom antiviral resistance testing was performed. We pooled data from national studies to identify possible risk factors for infection with a resistant virus and to ascertain whether such infections led to influenza illness of different severity. Information on demographic and clinical variables was obtained from patients or their physicians. Odds ratios for infection with an oseltamivir resistant virus and relative risks for developing certain clinical outcomes were computed and adjusted through multivariable analysis. Overall, 727 (24.3%) of 2,992 tested influenza A(H1N1) viruses from 22 of 30 European countries were oseltamivir-resistant. Levels of resistance ranged from 1% in Italy to 67% in Norway. Five countries provided detailed case-based data on 373 oseltamivir resistant and 796 susceptible cases. By multivariable analysis, none of the analysed factors was significantly associated with an increased risk of infection with anoseltamivir-resistant virus. Similarly, infection with an oseltamivir-resistant virus was not significantly associated with a different risk of pneumonia, hospitalisation or any clinical complication. The large-scale emergence of oseltamivir-resistant viruses in Europe calls for a review of guidelines for influenza treatment.

Myocardial kinetics of thallium-201 after dipyridamole infusion in normal canine myocardium and in myocardium distal to a stenosis.

The purpose of the present study was to define myocardial and blood thallium-201 (Tl-201) kinetics after infusion of dipyridamole in normal canine myocardium and in myocardium distal to a coronary artery stenosis. Miniature radiation detector probes were implanted in the left ventricle in 39 open-chest dogs. A balloon constrictor was placed around the proximal left circumflex coronary artery. Electromagnetic flow probes were positioned proximally around both the left circumflex and left anterior descending coronary arteries. In five control dogs (group 1) the balloon occluder was not inflated; in 12 dogs (group 2) a mild stenosis was created such that resting flow was not reduced, yet the hyperemic response after 10 s of total occlusion was partially attenuated; in nine dogs (group 3) a moderate stenosis was created such that resting flow was not reduced, yet the hyperemic response was completely eliminated; and in 13 dogs (group 4) a severe stenosis was created such that resting flow was reduced. After intravenous dipyridamole (0.08 mg/kg . min-1 x 4 min), 1.5 mCi Tl-201 was injected intravenously and probe counts were collected continuously for 4 h. The mean 4-h fractional myocardial Tl-201 clearance for nonstenotic zones was 0.35, 0.27 for group 2 stenotic zones, 0.19 for group 3 stenotic zones, and 0.05 for group 4 stenotic zones (P less than 0.0001). After reaching peak activity, myocardial Tl-201 activity cleared biexponentially with a final decay constant lambda 2 = 0.0017 +/- 0.0001 min-1 (SE) for nonstenotic zones, 0.0011 +/- 0.0001 min-1 for group 2 stenotic zones, and 0.0006 +/- 0.0001 min-1 for group 3 stenotic zones (P less than 0.01). Group 4 stenotic zone Tl-201 clearances were negligible (decay constant essentially zero). Blood Tl-201 activity decayed triexponentially with a final blood lambda 3 = 0.0018 +/- 0.0001 min-1, which was almost identical to the final myocardial lambda 2 decay constant. Thus, the rate of myocardial Tl-201 clearance can distinguish between coronary stenoses of graded hemodynamic severity. These results may be applicable to quantitative techniques for determining myocardial Tl-201 clearance rates on serial clinical images after dipyridamole administration.

Selective inhibition of syncytium-inducing and nonsyncytium-inducing HIV-1 variants in individuals receiving didanosine or zidovudine, respectively.

By studying changes in the clonal composition of HIV-1 populations during the first weeks of zidovudine (ZDV) treatment before the development of ZDV resistance-conferring mutations, we demonstrated previously a selective inhibition of nonsyncytium-inducing (NSI) HIV-1, even when present as coexisting population in individuals also harboring syncytium-inducing (SI) HIV-1. In this study, we observed the opposite in individuals receiving didanosine (ddI) treatment. In these individuals (n = 7) a median -0.98 log change (range -1.55-0.08) in infectious cellular SI load was observed, whereas the coexisting NSI load was only minimally affected (median -0.15 log, range -1.27-0.50; P = 0.03). The virus phenotype-dependent treatment responses were independent of the clonal composition of HIV-1 populations at baseline. Individuals treated with a combination of ZDV and ddI revealed an equal decline of both NSI and SI infectious cellular load (n = 4; NSI: median -1.55 log, range -2.19 to -1.45; SI: median -1.47 log, range -1.81 to -0.86; P = 0.56). To test the hypothesis that the previously reported optimal activation of ZDV and ddI in activated and resting T cells, respectively, in combination with the differential T cell tropism of NSI and SI HIV-1 is the basis for the observed virus phenotype specific efficacy of nucleoside analogs, we studied the effect of treatment with a protease inhibitor that does not require intracellular activation. Individuals receiving ritonavir (n = 4) indeed showed equal declines in NSI and SI infectious cellular load (NSI: median -2.37 log, range -2.59 to -2.16; SI: median -2.82 log, range -3.14 to -2.50; P = 0.25). Our data suggest HIV-1 phenotype as an additional parameter in the design of optimal treatment regimens.

[HIV should become a notifiable disease]. / Hiv moet meldingsplichtig worden.

In recent years, it has become evident that primary HIV infections are largely responsible for new cases. In order to identify the chains of transmission involved, HIV should become a notifiable disease in the Netherlands.

Ending the epidemic: Critical role of primary HIV infection.

Early identification and immediate treatment of individuals newly infected with HIV is important for two reasons: it benefits the long-term health of the infected patient, and it reduces onward HIV transmission. Primary HIV infection (PHI) reflects the period following HIV acquisition during which viraemia bursts until the establishment of a stable plasma HIV-RNA level approximately six months post infection. During this period, patients are particularly contagious and are often unaware of the infection. As a consequence, PHI disproportionally affects onward transmission. During PHI the immune system is irreparably damaged and persistent viral reservoirs are formed. Initiating antiretroviral therapy (ART) during PHI could potentially lead to a functional cure through early and prolonged viral suppression. Unfortunately, symptoms of PHI are nonspecific and the diagnosis is frequently missed. This impedes timely diagnosis and prompt initiation of ART. To increase awareness and underscore the importance of immediate ART initiation, we describe here the pathogenesis, clinical presentation, and impact of treating PHI.

A novel real-time PCR assay to determine relative replication capacity for HIV-1 protease variants and/or reverse transcriptase variants.

The emergence of drug-resistant viruses is a major issue in the treatment of HIV-1 infections. Quite often these drug-resistant viruses have a reduced replication capacity. A novel assay was developed to study the impact of mutations selected during therapy on viral replication capacity. Two HIV-1 HXB2 reference clones were constructed for this assay based on viral competition experiments, which are identical except for the presence of two silent nucleotide changes in p24 in one of the two clones. Within these two reference clones, three different contiguous deletions were constructed: (I) the C-terminus of Gag and protease, (II) the N-terminus of RT and (III) the C-terminus of Gag and protease together with the N-terminus of RT. Using these reference clones, recombinant viruses were created and viral competition experiments were performed. The proportion of each virus during the competition experiments was determined with a real-time PCR assay based on the two silent nucleotide changes in p24 in one of the two reference clones. With this novel assay it was possible to detect accurately differences in replication capacity due to mutations in the C-terminus of Gag and protease and/or the N-terminus of RT.

The novel activation of ABL by fusion to an ets-related gene, TEL.

In human leukemia, activation of the ABL proto-oncogene locus on chromosome 9 most commonly occurs as a result of its fusion to the BCR locus on chromosome 22. The resulting chimeric protein displays an elevated tyrosine kinase activity. We have identified a novel activation of ABL which involves a gene located on chromosome 12, designated TEL. Like BCR, TEL is fused in-frame with ABL and produces a fusion protein with an elevated tyrosine kinase activity when assayed in an immune complex. The amino-terminal sequences of TEL encode a helix-loop-helix motif which may mediate dimerization.

Quantitative split dose thallium-201 imaging with exercise: a technique for obtaining rest and exercise perfusion images in one setting and markedly reducing the study time.

In the current study, a technique for performing serial thallium imaging after two separate tracer injections was applied to exercise thallium imaging, thus allowing the acquisition of rest and exercise images within 1 hour. Twenty-four patients with and 10 patients without significant coronary artery disease were studied. One mCi of thallium-201 was injected intravenously and imaging was performed at rest in three projections. The patient was then stressed and an additional 1 mCi of thallium injected during exercise. Images in the same three projections were collected. After computer realignment, the rest image was subtracted from the exercise image to produce an image representing perfusion during exercise. All 24 patients with coronary artery disease had a positive study, while 9 of 10 without disease had a negative study. The images were then interpreted using a computer method designed to quantitate regional myocardial thallium distribution and redistribution. With quantitative interpretation, 23 of 24 patients with coronary disease had a positive study, while only 1 without disease had a positive study. With qualitative interpretation, 39 (89%) of 44 stenosed coronary arteries demonstrated thallium defects in corresponding myocardial segments, while 54 (93%) of 58 nonstenosed coronary arteries did not. With quantitative interpretation, 38 (86%) of 44 stenosed coronary arteries demonstrated thallium defects in corresponding myocardial segments, while 53 (91%) of 58 nonstenosed coronary arteries did not. A split dose thallium imaging technique that allows imaging before and immediately after exercise, thus markedly reducing the study time, has been validated.

Technetium-99m isonitrile myocardial uptake at rest. I. Relation to severity of coronary artery stenosis.

To determine the potential of planar technetium-99m methoxybutyl isonitrile myocardial imaging as a method of detecting totally occluded or severely stenosed coronary arteries, the regional distribution of technetium-99m isonitrile at rest was compared with the coronary anatomy in 38 patients with prior myocardial infarction who underwent coronary arteriography. Left ventricular technetium-99m isonitrile tracer uptake at rest was assessed in the three major coronary vascular territories. When qualitative rest technetium-99m isonitrile uptake was markedly reduced or absent (grade 0), there was a 91% probability of finding a totally occluded or severely stenosed coronary artery. When qualitative tracer uptake was reduced (grade 1) or normal (grade 2), it excluded all territories supplied by a totally occluded vessel with poor collateral flow. Quantitative technetium-99m isonitrile uptake (mean +/- 1 standard deviation) in territories supplied by an occluded coronary artery with poor collateral flow (42 +/- 21%) was lower than in territories supplied by a vessel with less than 50% stenosis (87 +/- 10%) and 50 to 99% stenosis (74 +/- 19%) (p less than 0.001). Furthermore, technetium-99m isonitrile uptake in areas supplied by an occluded coronary artery with good collateral flow (61 +/- 23%) was lower than in areas supplied by a vessel with less than 50% stenosis (87 +/- 10%) (p less than 0.001). Because rest technetium-99m isonitrile imaging detects coronary occlusion with poor collateral flow, this method may be useful in assessing patients with acute myocardial infarction.