Charles A. Boucher

Drug-resistance genotyping in HIV-1 therapy: the VIRAD APT randomi sed controlled trial

BACKGROUND Growing evidence has linked HIV-1 resistance mutations and drug failure. The use of genotypic-resistance analysis to assist therapeutic decision-making in patients failing therapy has not been investigated. We assessed the virological and immunological impact of genotypic-resistance testing. METHODS We did a prospective, open, randomised, controlled study of HIV-1-infected patients in whom combination therapy was not successful. We randomly assigned patients standard care (control, n=43) or treatment according to the resistance mutations in protease and reverse-transcriptase genes (genotypic group, n=65). The major endpoint was the change in HIV-1 RNA viral load. Analysis was by intention to treat. FINDINGS 108 patients were enrolled. All patients were similar for risk factors, age, sex, previous treatment, CD4-cell count (214/microL [SD14]) and log HIV-1 RNA viral load at baseline (4.7 copies/mL [0.1]). At month 3, the mean change in HIV-1 RNA was -1.04 log (0.14) in the study group compared with -0.46 log (0.17) in the control group (mean difference 0.58 log [95% CI 0.14-1.02], p=0.01). At month 6, changes were -1.15 (0.15) log copies/mL, and -0.67 (0.19) log copies/mL in the genotypic group and the control group, respectively (mean difference 0.48 log [0.01-0.97], p=0.05). Difference in the drop in viral load combined at 3 months and 6 months was significant (p=0.015). At month 3, HIV-1 RNA was lower than detection level (200 copies/mL) in 29% (19/65) of patients in the genotypic group versus 14% (6/43) in the control group (p=0.017). At month 6, the values were 32% (21/65) and 14% (6/43) (p=0.067) for the genotypic group and the control group, respectively. Therapy was generally well tolerated, with ten patients (six in the genotypic group, four in the control group) requiring toxic-effect-related drug modification. INTERPRETATION We found genotypic-resistance testing to have a significant benefit on the virological response when choosing a therapeutic alternative. Further study of the use of genotypic-resistance testing in assisting clinical decision-making is warranted.

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.

Determination of Cardiac Risk by Dipyridamole-Thallium Imaging before Peripheral Vascular Surgery

To evaluate the severity of coronary artery disease in patients with severe peripheral vascular disease requiring operation, we performed preoperative dipyridamole-thallium imaging in 54 stable patients with suspected coronary artery disease. Of the 54 patients, 48 had peripheral vascular surgery as scheduled without coronary angiography, of whom 8 (17 per cent) had postoperative cardiac ischemic events. The occurrence of these eight cardiac events could not have been predicted preoperatively by any clinical factors but did correlate with the presence of thallium redistribution. Eight of 16 patients with thallium redistribution had cardiac events, whereas there were no such events in 32 patients whose thallium scan either was normal or showed only persistent defects (P less than 0.0001). Six other patients also had thallium redistribution but underwent coronary angiography before vascular surgery. All had severe multivessel coronary artery disease, and four underwent coronary bypass surgery followed by uncomplicated peripheral vascular surgery. These data suggest that patients without thallium redistribution are at a low risk for postoperative ischemic events and may proceed to have vascular surgery. Patients with redistribution have a high incidence of postoperative ischemic events and should be considered for preoperative coronary angiography and myocardial revascularization in an effort to avoid postoperative myocardial ischemia and to improve survival. Dipyridamole-thallium imaging is superior to clinical assessment and is safer and less expensive than coronary angiography for the determination of cardiac risk.

Drug Resistance Mutations for Surveillance of Transmitted HIV-1 Drug-Resistance: 2009 Update

Programs that monitor local, national, and regional levels of transmitted HIV-1 drug resistance inform treatment guidelines and provide feedback on the success of HIV-1 treatment and prevention programs. To accurately compare transmitted drug resistance rates across geographic regions and times, the World Health Organization has recommended the adoption of a consensus genotypic definition of transmitted HIV-1 drug resistance. In January 2007, we outlined criteria for developing a list of mutations for drug-resistance surveillance and compiled a list of 80 RT and protease mutations meeting these criteria (surveillance drug resistance mutations; SDRMs). Since January 2007, several new drugs have been approved and several new drug-resistance mutations have been identified. In this paper, we follow the same procedures described previously to develop an updated list of SDRMs that are likely to be useful for ongoing and future studies of transmitted drug resistance. The updated SDRM list has 93 mutations including 34 NRTI-resistance mutations at 15 RT positions, 19 NNRTI-resistance mutations at 10 RT positions, and 40 PI-resistance mutations at 18 protease positions.

Pathogenesis and transmission of swine-origin 2009 A(H1N1) influenza virus in ferrets

“Swine Flu” Pathology The clinical spectrum of disease caused by the swine-origin 2009 A(H1N1) influenza virus and its transmissibility are not completely understood. Munster et al. (p. 481; published online 2 July) and Maines et al. (p. 484; published online 2 July) used ferrets, an established model for human influenza, to evaluate the pathogenesis and transmissibility of a selection of 2009 A(H1N1) virus isolates compared with representative seasonal H1N1 viruses. The results help explain the atypical symptoms seen so far, including the gastrointestinal distress and vomiting observed in many patients. Although results were variable, it seems that the 2009 A(H1N1) virus may be less efficiently transmitted by respiratory droplets in comparison to the highly transmissible seasonal H1N1 virus, suggesting that additional virus adaptation in mammals may be required before we see phenotypes observed in earlier pandemics. Animal experiments compare the dynamics and effects of the virus causing the 2009 flu outbreak to those of seasonal H1N1 flu. The swine-origin A(H1N1) influenza virus that has emerged in humans in early 2009 has raised concerns about pandemic developments. In a ferret pathogenesis and transmission model, the 2009 A(H1N1) influenza virus was found to be more pathogenic than a seasonal A(H1N1) virus, with more extensive virus replication occurring in the respiratory tract. Replication of seasonal A(H1N1) virus was confined to the nasal cavity of ferrets, but the 2009 A(H1N1) influenza virus also replicated in the trachea, bronchi, and bronchioles. Virus shedding was more abundant from the upper respiratory tract for 2009 A(H1N1) influenza virus as compared with seasonal virus, and transmission via aerosol or respiratory droplets was equally efficient. These data suggest that the 2009 A(H1N1) influenza virus has the ability to persist in the human population, potentially with more severe clinical consequences.

An automated genotyping system for analysis of HIV-1 and other microbial sequences

MOTIVATION Genetic analysis of HIV-1 is important not only for vaccine development, but also to guide treatment strategies, track the emergence of new viral variants and ensure that diagnostic assays are contemporary and fully optimized. However, most genotyping methods are laborious and complex, and involve the use of multiple software applications. Here, we describe the development of an automated genotyping system that can be easily applied to HIV-1 and other rapidly evolving viral pathogens. RESULTS The new REGA subtyping tool, developed using Java programming and PERL scripts, combines phylogenetic analyses with boot-scanning methods for the genetic subtyping of full-length and subgenomic fragments of HIV-1. When used to investigate the subtype of previously published reference datasets that were analysed using manual phylogenetic methods, the automated method correctly identified 97.5-100% of non-recombinant and circulating recombinant forms of HIV-1, including 108 full-length, 108 gag and 221 env sequences downloaded from the Los Alamos database.

Prevalence of Drug-Resistant HIV-1 Variants in Untreated Individuals in Europe: Implications for Clinical Management

BACKGROUND Infection with drug-resistant human immunodeficiency virus type 1 (HIV-1) can impair the response to combination therapy. Widespread transmission of drug-resistant variants has the disturbing potential of limiting future therapy options and affecting the efficacy of postexposure prophylaxis. METHODS We determined the baseline rate of drug resistance in 2208 therapy-naive patients recently and chronically infected with HIV-1 from 19 European countries during 1996-2002. RESULTS In Europe, 1 of 10 antiretroviral-naive patients carried viruses with > or = 1 drug-resistance mutation. Recently infected patients harbored resistant variants more often than did chronically infected patients (13.5% vs. 8.7%; P=.006). Non-B viruses (30%) less frequently carried resistance mutations than did subtype B viruses (4.8% vs. 12.9%; P<.01). Baseline resistance increased over time in newly diagnosed cases of non-B infection: from 2.0% (1/49) in 1996-1998 to 8.2% (16/194) in 2000-2001. CONCLUSIONS Drug-resistant variants are frequently present in both recently and chronically infected therapy-naive patients. Drug-resistant variants are most commonly seen in patients infected with subtype B virus, probably because of longer exposure of these viruses to drugs. However, an increase in baseline resistance in non-B viruses is observed. These data argue for testing all drug-naive patients and are of relevance when guidelines for management of postexposure prophylaxis and first-line therapy are updated.

Reduced replication of 3TC-resistant HIV-1 variants in primary cells due to a processivity defect of the reverse transcriptase enzyme.

Human immunodeficiency virus type 1 (HIV‐1) variants with resistance mutations in the reverse transcriptase (RT) gene appear during drug therapy with the nucleoside analogue 2′,3′‐dideoxy‐3′‐thiacytidine (3TC). These resistance mutations alter the methionine (Met) residue of the conserved YMDD motif, which is part of the catalytic core of the RT enzyme. Isoleucine (Ile) variants are initially observed, followed by the appearance and eventual outgrowth of viruses encoding valine (Val). Similar replication kinetics were measured for wild‐type and 3TC‐resistant HIV‐1 viruses in tissue culture infections of a T cell line, but we measured reduced polymerase activity for the two mutant RT enzymes compared with the wild‐type enzyme (Ile = 43% and Val = 67%). Gel analysis of the reverse transcription products revealed that both 3TC‐resistant RT mutants produce significantly shorter cDNA molecules than the wild‐type enzyme [Met (wt)>Val>Ile], indicating that 3TC‐resistant RT polymerases are less processive enzymes. Interestingly, these enzyme defects were more pronounced under limiting dNTP concentrations and we therefore assayed virus replication in primary cells that contain relatively low dNTP levels. Under these conditions, we measured significantly reduced replication kinetics for the 3TC‐resistant HIV‐1 variants [Met (wt)>Val>Ile]. If the level of virus replication can be similarly reduced in 3TC‐treated patients that develop drug‐resistant HIV‐1 variants, this may be of considerable clinical benefit.

Increased fitness of drug resistant HIV-1 protease as a result of acquisition of compensatory mutations during suboptimal therapy.

OBJECTIVE It is thought as a consequence of continuous replication, HIV-1 has acquired an optimal fitness state and that suboptimal antiretroviral therapy selects for drug resistant variants which show impaired fitness in the absence of the drug. In this paper we studied the evolution and fitness of viral populations appearing in a patient who received protease monotherapy. METHODS Two factors contributing to fitness, drug resistance and protease catalytic activity, were studied at the enzymatic and virological level. RESULTS The first drug resistant viral variants that were selected in vivo harboured one to three protease substitutions. These mutants showed reduced protease activity and consequently a reduction in viral replication capacity. During continued in vivo replication of these viruses in the presence of the drug, novel variants harbouring additional substitutions in the viral protease appeared. These variants did not display any further increase in drug resistance but demonstrated clearly increased protease activity. Consequently the replication capacity of these viruses was raised to a level at which they replicated better than the original wild-type virus. CONCLUSION This study indicates that the viral population in the patient does not have to represent the fittest possible variants, and thus antiretroviral therapy may drive the viral population first through a lower fitness level and then to a higher fitness level.

Serial thallium-201 myocardial imaging after dipyridamole infusion: diagnostic utility in detecting coronary stenoses and relationship to regional wall motion.

After a 4-minute i.v. dipyridamole infusion, 0.14 mg/kg/min, serial thallium-201 scans were obtained in 60 patients undergoing cardiac catheterization. Forty patients had significant (> 50% stenosis) coronary artery disease (CAD), and 20 patients had normal coronary arteries or trivial lesions. The images were graded qualitatively for thallium activity by three observers. Sensitivity was 93% (37 of 40) and specificity was 80% (16 of 20). The sensitivity and specificity of the thallium-201 study were not affected by the extent of CAD, the presence of Q waves, or propranolol therapy. Twenty-seven of 37 patients who had initial defects (73%) had complete thallium redistribution of one or more defects. Patient-by-patient analysis using a regression model of all patients showed that the fate of a segmental thallium defect predicted abnormal wall motion by angiography better than ECG Q waves. The presence of propranolol therapy or collaterals did not significantly affect the thallium redistribution results.We conclude that qualitative interpretation by multiple observers of thallium images after dipyridamole infusion is a highly sensitive and specific test for CAD. After dipyridamole, as with exercise stress, the extent of thallium redistribution is related to the degree of myocardial wall motion abnormality.