ports. A survey in our laboratory of viruses containing L90M from 78 patient samples demonstrates this point even more clearly (figure 1). The data clearly demonstrate the limitations of genotypic analyses that rely on identification of known mutations, since a significant proportion (40%) of viruses containing the L90M “resistance-associated” mutation remained sensitive to saquinavir. The additional genetic changes that modulate the effects of L90M on protease inhibitor resistance are not known. Dr. Bakker criticizes our work as anecdotal and likely to fuel the debate over the relative merits of phenotyping versus genotyping. The characterization of drug susceptibility patterns in these 2 patients, who, to our knowledge, were the first previously treatment-naive patients to experience virus load rebound while undergoing triple combination therapy, is indeed anecdotal. However, strikingly similar results have subsequently been described in larger studies, although not in as much detail on an individual patient basis [6, 7]. The purpose of our study was to carefully characterize the evolution of viral drug susceptibility in a small number of patients treated in an early triple combination drug trial and to generate hypotheses about viral drug resistance for testing in larger clinical trials. The large-scale clinical trials Dr. Bakker calls for are under way, but results are still many months to years away. Until then, detailed but small-scale and anecdotal studies can provide valuable preliminary data to augment our understanding of HIV drug resistance. Finally, Dr. Bakker correctly points out that the phenotypic assay we developed, as well as other phenotypic assays [8, 9] and most genotypic assays, has a limited ability to detect minor populations of drug-resistant virus. Traditional recombinant virus phenotypic assays that rely on the outgrowth of replication-competent HIV-1 from transfected DNA fragments, such as that cited by Dr. Bakker [10], are especially subject to this limitation, because less fit viruses may be underrepresented in the virus stock used for the susceptibility assay. The assay used in the present study [11] does not involve culture of replication-competent virus and therefore is less subject to this potential bias. Nonetheless, we agree with the statement that phenotypic and genotypic assays are complementary and valuable tools; the concluding statement in our paper reflects this viewpoint.

The HIV protease inhibitor P-1946 is a member of a novel family of l-Lysine derivatives. The compound is a specific HIV-1 protease inhibitor that has potent and selective in vitro antiviral activity (EC50 152 nM) against a range of isolates resistant to commercially available protease inhibitors. The presence of at least four primary and four secondary drug resistance mutations is required to achieve greater than four-fold resistance to P-1946. P-1946's favorable resistance profile makes it a good lead for the development of new agents active against existing PI-resistant virus in treatment-experienced patient.

Although suppression of HIV-1 RNA below the limit of detection is associated with optimal outcomes, many patients can maintain or increase their CD4(+) count for prolonged time periods in the presence of persistent low-level viremia. We followed seven patients with prolonged (>5 years) discordant CD4(+)/viral load (VL) responses on protease inhibitor (PI)-based highly active antiretroviral therapy (HAART) prospectively for 1 year to assess evolution of immune function, viral phenotype, replication capacity (RC), and resistance profile. Immune function was assessed by qualitative and quantitative measurement of cellular activation (CD38(+)HLA-DR(+) and CD38 antibodies bound per cell), and the interferon (IFN)-() ELISpot assay. Presence of syncytium-inducing (SI) or nonsyncytium-inducing (NSI) viral strains was determined by MT-2 cell culture. RC was measured by a modified rapid recombinant virus assay. The resistance profile was characterized by both genotypic and phenotypic analysis. Over the year of follow-up, IFN-() production to gag persisted, responses to other HIV antigens increased, and markers of cellular activation did not change. NSI virus predominated. The genotypic (GSS) and phenotypic (PSS) susceptibility scores remained stable. Evolution of RC was variable over the year of follow-up, but the RC of viruses remained well below that of wild-type clinical isolates. Thus, CD4(+)/VL discordance can be maintained for periods exceeding 5 years in some patients receiving PI-based HAART without significant evolution of HIV resistance.

Objective: To determine the influence of pol replication capacity on the natural history of HIV-1 infection. Design: Pol replication capacity was measured using a recombinant virus single cycle assay on baseline plasma specimens from subjects enrolled in the Hemophilia Growth and Development Study. Setting: Children and adolescents with hemophilia and HIV-1 infection were enrolled at multiple sites across the USA into a natural history study. Participants: The Hemophilia Growth and Development Study enrolled 207 HIV-1-infected hemophiliacs between 6 and 19 years of age in 1989 and 1990. Subjects were followed every 6 months through 1997 with pol replication capacity measurements available from 128 of the subjects. Main outcome measures: A univariate model defined the relationship between pol replication capacity and HIV-1 RNA and CD4 T-cell number. A random effects model assessed the ability of this measure to predict CD4 T-cell decline over time and a Cox proportional hazards model and Kaplan–Meier analyses defined how it predicts clinical progression. Results: Pol replication capacity measures correlated with baseline HIV-1 RNA, R2 = 0.189 (P = 0.03) and CD4 T-cell number, −0.197 (P = 0.03). It also independently predicted CD4 T-cell decline over time and progression to AIDS. Conclusions: This study demonstrates that pol replication capacity independently influences the natural history of HIV-1 infection.

Objective: To assess phenotype susceptibility testing (PHENO) with standard of care (SOC) to improve antiretroviral therapy. Design: A prospective, multicenter study of 238 patients taking a stable antiretroviral regimen for > 6 months, with one or two protease inhibitors (PI) and entry HIV RNA > 400 copies/ml.Method: Patients were randomized to receive or not receive PHENO results for selecting antiretroviral regimens. Primary outcome was HIV RNA measures.Results: At baseline, median CD4 cell count was 277 x 10(6) cells/I and HIV RNA was 10 000 copies/ml; 76% had not taken a non-nucleoside reverse transcriptase inhibitor drug (NNRTI). There were significant differences between the groups in selection of baseline nucleoside reverse transcriptase inhibitor (NRTI). At month 6, reduction in HIV RNA was 0.71 and 0.69 log(10) copies/ml for PHENO and SOC, respectively; the proportion with < 400 copies/ml (48%) was the same for both groups. No differences were seen at month 12. In a subgroup with resistance to four or more PI, 50% of the PHENO versus 17% of the SOC had HIV RNA < 400 copies/ml at month 6 (P = 0.02). The number of NNRTI and PI, but not NRTI, in the regimen that were active by phenotype at baseline was a strong independent predictor of viral suppression (P < 0.006). Use of alternative NRTI sensitivity cut-offs improved their predictive value.Conclusions: Although virological outcome was similar in both groups, the potential benefit of PHENO was seen in patients with more resistant virus. Lack of appropriate cut-offs may have partially accounted for the lack of benefit from PHENO and demonstrated the need to identify clinically relevant sensitivity cut-off points. (c) 2005 Lippincott Williams C Wilkins

ABSTRACTWild-type viruses from the ViroLogic phenotype-genotype database were evaluated to determine the upper confidence limit of the drug susceptibility distributions, or “biological cutoffs,” for the PhenoSense HIV phenotypic drug susceptibility assay. Definition of the natural variation in drug susceptibility in wild-type human immunodeficiency virus (HIV) type 1 isolates is necessary to determine the prevalence of innate drug resistance and to assess the capability of the PhenoSense assay to reliably measure subtle reductions in drug susceptibility. The biological cutoffs for each drug, defined by the 99th percentile of the fold change in the 50% inhibitory concentration distributions or the mean fold change plus 2 standard deviations, were lower than those previously reported for other phenotypic assays and lower than the clinically relevant cutoffs previously defined for the PhenoSense assay. The 99th percentile fold change values ranged from 1.2 (tenofovir) to 1.8 (zidovudine) for nucleoside reverse transcriptase RT inhibitors (RTIs), from 3.0 (efavirenz) to 6.2 (delavirdine) for nonnucleoside RTIs, and from 1.6 (lopinavir) to 3.6 (nelfinavir) for protease inhibitors. To evaluate the potential role of intrinsic assay variability in the observed variations in the drug susceptibilities of wild-type isolates, 10 reference viruses with different drug susceptibility patterns were tested 8 to 30 times each. The median coefficients of variation in fold change for the reference viruses ranged from 12 to 18% for all drugs except zidovudine (32%), strongly suggesting that the observed differences in wild-type virus susceptibility to the different drugs is related to intrinsic virus variability rather than assay variability. The low biological cutoffs and assay variability suggest that the PhenoSense HIV assay may assist in defining clinically relevant susceptibility cutoffs for resistance to antiretroviral drugs.

We analyzed retrospectively, by use of logistic regression, the role of the phenotypic susceptibility score (PSS) as a determinant of virologic outcome in a study of treatment-experienced human immunodeficiency virus (HIV) type 1-infected subjects. A model for PSS in which each drug was treated equally and scored as active or inactive was not predictive of HIV-1 RNA load (VL) suppression. However, weighting the potential contribution of each drug on the basis of inferred potency and by use of a continuous scale to quantify drug susceptibility revealed significant associations between PSS and outcome. Entry VL was a strong independent predictor of therapy outcome, and PSS was a strong predictor of the magnitude of the initial decrease in VL. This suggests that the prospective evaluation of PSS to identify regimens that maximize decreases in VL to reduce the probability of virologic rebound could improve antiretroviral treatment.

Background: Antiretroviral therapy has dramatically reduced the morbidity and mortality of infection due to HIV. The emergence of drug-resistant virus has limited the usefulness of many drugs. Objective: To determine the prevalence of HIV drug resistance in the population of adults receiving care in the United States. Design and methods: HIV drug susceptibility assays were performed on plasma virus from a random sample representative of the 132 500 HIV-infected American adults who had received medical care in early 1996 yet were viremic with > 500 copies/ml of HIV RNA in late 1998. A blood sample was obtained from 1797 patients who comprised a representative sample of the 208 900 adults receiving urban care for HIV infection in early 1996 who survived to late 1998. The sampling procedure permitted weighting each evaluated patient to reflect demographic and other characteristics of the target population. Results: We estimated that 132 500 (63%) of the target population had HIV viremia of > 500 copies/ml. Among viremic patients, an estimated 76% had resistance to one or more antiretroviral drugs. The odds of resistance were significantly higher in patients with a history of antiretroviral drug use, advanced HIV disease, higher plasma HIV viral load and lowest CD4 cell count by self-report. Conclusions: The frequent selection for drug-resistant virus among viremic patients during the first 3 years of widespread use of potent antiretroviral combination therapy has significant implications for HIV treatment and transmission.

In most European countries, HIV drug resistance testing has become a routine clinical tool. However, its practical implementation in a clinical context is demanding. The European HIV Drug Resistance Panel was established to make recommendations to clinicians and virologists on this topic and to propose quality control measures. The panel recommends resistance testing for the following indications: i) drug-naive patients with acute or recent infection; ii) therapy failure, including suboptimal treatment response, when treatment change is considered; iii) pregnant HIV-1-infected women and paediatric patients with detectable viral load when treatment initiation or change is considered; and iv) genotype source patient when post-exposure prophylaxis is considered. In addition, for drug-naive patients with chronic infection in whom treatment is to be started, the panel suggests that resistance testing should be strongly considered and recommends testing the earliest sample for drug resistance if suspicion of resistance is high or prevalence of resistance in this population exceeds 10%. The panel does not favour genotyping over phenotype, however it is anticipated that genotyping will be used more often because of its greater accessibility, lower cost and faster turnaround time. For the interpretation of resistance data, clinically validated systems should be used to the greatest extent possible. It is mandatory that laboratories performing HIV resistance tests take regular part in quality assurance programs. Similarly, it is necessary that HIV clinicians and virologists take part in continuous education and meet regularly to discuss problematic clinical cases. Indeed, resistance test results should be used in the context of all other clinically relevant information for predicting therapy response. The panel also encourages the timely collection of epidemiological information to estimate the impact of transmission of resistant HIV and the prevalence of HIV-1 non-B subtypes in the different European countries.

Qari and colleagues (3) in 2002 reported a comparative analysis of two commercial phenotypic assays (Antivirogram and PhenoSense) for human immunodeficiency virus type 1 (HIV-1) drug resistance testing. The Antivirogram (1) and PhenoSense (2) assays are both recombinant virus-based assays to measure the drug susceptibilities of patient-derived HIV protease or reverse transcriptase sequences and were developed by Virco (Belgium) and ViroLogic, Inc. (South San Francisco, Calif.), respectively. In the paper, the authors collected viruses from 50 clinical specimens and measured their susceptibility to 12 to 15 antiretroviral drugs using both assays. Based on 529 pairs of observations, they reported a 91.5% concordance by binary classification of sensitive versus reduced susceptibility. They concluded that “the Antivirogram and PhenoSense assays correlate well, despite the use of different testing strategies.” Because of the lack of quantitative data in their paper on the actual correlation coefficients for the two phenotypic assays, we downloaded resistance estimates (increases [n-fold] in 50% inhibitory concentrations relative to that for the wild type) for the samples used by Qari et al. (3) from the Stanford HIV drug resistance database (4). Despite the high binary concordance obtained with the samples that they tested, correlations between the resistance values were surprisingly poor for many of the drugs (Table ​(Table1).1). For some drugs, such as delavirdine (DLV), the two assays had high Pearson correlation coefficients, but these high correlations were due to a small number of highly resistant samples. We have focused, therefore, on Spearman rank correlation coefficients (rs) since they are less sensitive to extreme values. For the 14 drugs, rs values ranged from 0.018 for saquinavir (SQV) to 0.551 for lamivudine (3TC), with a median of 0.290. Spearman's rank correlation test is a statistical test in which the null hypothesis is that two variables are uncorrelated. Performing a separate test for each drug, we failed to reject this null hypothesis for eight drugs using a significance value of 0.05 (Table ​(Table11). TABLE 1. Correlation between Antivirogram and PhenoSense phenotypic assays for 14 antiretroviral drugs Furthermore, from Table ​Table11 of the original paper, we noticed that only a small fraction of the samples were classified as having “reduced susceptibility” by both assays. We argue that because of the high fraction of drug-susceptible viruses in their study, their overall 91.5% concordance result is somewhat misleading. (Given frequencies of susceptible viruses of 89.6 and 91.7% for the Antivirogram and PhenoSense assays, respectively, one would expect 83.0% concordance by chance). In clinical settings it is most important to have high prediction accuracy when the virus has reduced susceptibility to drugs. This argues for a closer examination of samples predicted to have reduced susceptibility on the basis of Qari et al.'s cutoff values (3). Of the 44 samples that are classified as having reduced susceptibility by the Antivirogram assay, 17 are reported as sensitive by the PhenoSense assay, resulting in a concordance of only 61.4% (95% confidence interval [CI], 45.5 to 75.6%). Of the 55 samples that are classified as having reduced susceptibility by the PhenoSense assay, 28 are reported as sensitive by the Antivirogram assay, resulting in a concordance of only 49.1% (95% CI, 35.4 to 62.9%). We conclude that resistance estimates from the Antivirogram and PhenoSense assays do not correlate well for the data shown by Qari et al. (3). Although these assays may correlate better for data sets that include more resistant viruses, in the absence of data showing that one test is better than the other, we suggest that classifications of reduced susceptibilities resulting from these phenotypic assays be interpreted with caution.

Background: Cross resistance is common among the non-nucleoside reverse transcriptase inhibitors (NNRTIs). G190A appears in 5-15% of the patients treated with nevirapine or efavirenz who develop clinical resistance. Objectives: In this study we investigated the effect of G190A and other NNRTI substitutions on the phenotypic susceptibility to this class of drugs. Study design: We identified 15 individuals, who after treatment with NNRTIs (nevirapine or efavirenz; median exposure of 20 months), developed isolated G190A, G190A in combination with K103N, or K103N alone. Phenotypic and genotypic analyses of stored plasma specimens were performed before and after the mutations occurred to assess NNRTI susceptibility. Results: All isolates that developed only G190A substitution became less susceptible to nevirapine (median: 125-fold) and efavirenz (median: 10-fold) but were 2.5-fold more sensitive to delavirdine (Wilcoxon P = 0.06). In the group with only K103N substitution, acquisition of resistance to all NNRTIs was observed. In the group with the double substitutions, G190A and K103N, delavirdine susceptibility decreased 13-fold, while resistance to nevirapine and efavirenz decreased by 239- and 154-folds, respectively (Kruskal-Wallis H P = 0.009). Conclusions: The data suggest that the presence of a G190A substitution attenuates the phenotypic resistance associated with a K103N substitution, although resistance is still present. The in vivo significance of the increased phenotypic susceptibility to delavirdine is not known but could be evaluated in a clinical trial. (C) 2004 Elsevier B.V. All rights reserved.

We evaluated phenotypic and genotypic markers of drug resistance in human immunodeficiency virus type 1 (HIV-1) at the time of virologic failure (VF) in subjects in the AIDS Clinical Trials Group Protocol 388 (ACTG 388) who received lamivudine-zidovudine (or lamivudine-stavudine) and either indinavir, efavirenz-indinavir, or nelfinavir-indinavir. At VF, phenotypically susceptible HIV-1 was found in 55% of subjects in the nelfinavir-indinavir arm, compared with 22% in the indinavir arm (P = .006). Phenotypic resistance to lamivudine was less common in the efavirenz-indinavir arm (33% of subjects; P = .002) and the nelfinavir-indinavir arm (43%; P = .003), compared with the indinavir arm (78%). Isolated phenotypic resistance to efavirenz at VF occurred in HIV-1 recovered from 33% of subjects in the efavirenz-indinavir arm; 24% of the subjects had HIV-1 with both efavirenz and lamivudine resistance. Results of genotypic tests were similar. The lower frequency of resistance in the nelfinavir-indinavir arm likely reflects decreased drug exposure that is due to intolerance, which is consistent with the lower potency and tolerability of this combination in ACTG 388. The lower frequency of lamivudine resistance in the efavirenz-indinavir arm is consistent with reports in other studies of potent regimens. Thus, although dual resistance to efavirenz and lamivudine occurred at VF in the efavirenz-indinavir arm, this risk was relatively low when evaluated in the context of the potency and tolerability of this regimen.

ABSTRACT The initial virus strains from as many as 12% of individuals with primary human immunodeficiency virus (HIV) infection have a 50% inhibitory concentration ≤0.4-fold that of HIV type 1NL4-3 (HIV-1NL4-3) to ritonavir (hypersusceptibility [HS]). There is also substantial variation in replicative capacity (RC) or an in vitro assay of the contributions of protease (PR) and reverse transcriptase to viral fitness. In chronically infected antiretrovirally treated patients, amprenavir HS has been associated with the mutation N88S in PR, but this mutation is not seen in untreated patients. In this study, virus strains from 182 cases of primary HIV infection were analyzed, and a highly significant association between HS and low RC (≤10% that of HIV-1NL4-3) was observed (P < 10−6). Multivariate analysis was used to determine the genotypic basis of ritonavir HS, analyzing all polymorphic amino acid sites and insertions from p7gag through PR. Decision tree models developed on the entire Gag-plus-PR data set and on PR alone gave overall correct classifications of 73 and 72%, respectively, on cross-validation. They were also able to predict low RC, with sensitivities of 69 and 62% and specificities of 84 and 70%, respectively. The analysis shows that ritonavir HS in untreated primary HIV infection is not associated with single mutations but with combinations of amino acids at polymorphic sites and that the same genotypes which confer HS to PR inhibitors confer low RC. This supports the view that variation in PR function is directly responsible for variation in fitness among strains in primary infection.

BACKGROUND:Infection with primary drug-resistant human immunodeficiency virus type 1 (HIV-1) has been associated with higher CD4(+) T cell counts in drug-naive patients, suggesting that altered viral pol replication capacity (RC) associated with drug resistance diminishes immune injury in vivo, independent of exposure to drugs.METHODS:Virus replication over a single cycle was measured by use of a viral test vector containing patient-derived HIV-1 protease and reverse transcriptase gene segments.RESULTS:Among 191 recently infected patients, pol RC ranged widely, with only 6% of the variance explained by drug-resistance mutations. Patients infected with a virus with a low pol RC (</=43% of the reference virus) had significantly higher CD4(+) T cell counts at study entry, independent of drug resistance and plasma HIV-1 RNA level, and over time, both before and during combination antiretroviral therapy.CONCLUSIONS:Viral pol RC may influence HIV-1 disease progression by affecting the amount and tissue distribution of viral replication. The pol RC value of 43% may represent a threshold below which HIV-1 has lowered virulence and is less able to deplete CD4(+) T cell counts.

Phenotypic assays measure the susceptibility of HIV-1 to antiretroviral drugs, and are used to guide treatment regimen choices. Analyses of baseline predictors of virological failure support the use of a 0.4-fold cutpoint for defining increased susceptibility (hypersusceptibility) to efavirenz in the PhenoSense HIV assay. The enhancement of the virological activity of efavirenz, associated with resistance to reverse transcriptase inhibitors, provides an opportunity to consider mutational and drug interactions in optimizing treatment.

Objective: To evaluate phenotypic drug susceptibility and non-nucleoside reverse transcriptase inhibitor hypersusceptibility as predictors of the time to virological failure. Design: In a randomized clinical trial, phenotypic susceptibility was retrospectively determined among 131 exclusively nucleoside reverse transcriptase inhibitor (NRTI)-experienced patients with baseline HIV-RNA levels greater than 2000 copies/ml. Subjects were assigned two NRTI drugs and were randomly assigned to nelfinavir, efavirenz, or both. Virological failure was defined as two HIV-RNA measurements of 2000 copies/ml or greater at or after week 16 and before treatment discontinuation. Methods: Using biological cut-offs to define resistance, assigned NRTI and randomized drug regimens, continuous and dichotomous phenotypic susceptibility scores (PSS) were calculated for each virus. Efavirenz hypersusceptibility as a dichotomous value was defined as less than 0.4-fold resistance. Associations between virological failure and continuous and dichotomous PSS were evaluated using Kaplan–Meier curves and Cox proportional hazards regression models. Results: A higher baseline viral load (P < 0.02) and lower dichotomous or continuous baseline PSS (P = 0.004 and P < 0.001, respectively) were independently associated with virological failure. In the 85 subjects who received efavirenz, efavirenz hypersusceptibility (P = 0.042, hazard ratio 0.43, 95% confidence interval 0.19–0.97) was independently associated with a reduced risk of virological failure. Conclusion: Reduced phenotypic susceptibility was a significant independent risk factor for virological failure. The presence of efavirenz hypersusceptibility appeared to enhance virological responses during treatment with efavirenz in combination with NRTIs. The retrospective calculation of continuous PSS accurately identified treatment regimens containing sufficient drug activity to prevent virological failure.

To review recent clinical data regarding viral fitness and its potential utility in clinical practice. Therapeutic failure of antiretroviral regimens is often due to the development of drug resistance. The viral loads achieved by these drug-resistant variants frequently remain suppressed below pretreatment levels. Furthermore, many patients with virologic failure continue to maintain stable CD4 counts and remain clinically well. One possible explanation for these observations is that the resistant virus that emerges during failure of highly active antiretroviral therapy is less fit than its wild-type counterpart as a result of the requirement to maintain resistance mutations that allow it to replicate in the presence of antiretroviral drugs. Since there are many patients with limited treatment options due to extensive resistance or drug toxicities, or both, there is intense interest in exploring the possibility that viral fitness can be exploited for clinical benefit. This review describes the assays that are used to measure fitness and replication capacity, and investigations of their utility in clinical practice.A number of methods have been developed to measure viral fitness. Replication kinetic and competitive culture assays are accurate, but labour and time intensive. The availability of a rapid, single-cycle recombinant assay that measures viral replication capacity presents the possibility that assessments of viral fitness could be incorporated into clinical management. The replication capacity assay appears to correlate with more standard measures of viral fitness, and recently accumulated data in both wild-type and drug resistant viral populations suggest that replication capacity describes an intrinsic characteristic of HIV-1. A number of investigations of the clinical utility of fitness and replication capacity assays have established correlations between these measurements and virologic and immunologic outcomes.Much progress has been made in the past year in our understanding of viral fitness and replication capacity. Assays that measure these viral characteristics have the potential to expand the range of clinical strategies employed against HIV-1. Further investigations are needed to firmly establish the clinical utility of these assays.

We have investigated a potential transmission chain of HIV-1 with drug resistance-associated mutations between three individuals over a period of 5 years by use of cloning and sequencing of viral genes, and phenotypic characterization. Viruses containing reverse transcriptase drug resistance-associated mutations were transmitted sequentially between three homosexual men (A, B, and C), and persisted in one individual for at least 4 years, despite intermittent therapy and reduced viral replicative capacity compared with wild-type strains. Clonal analysis of the envelope gene from semen and blood virus showed that the virus transmitted to patient C was more closely related to virus from the semen than the blood of patient B. Our data suggest that HIV variants with drug resistance-associated mutations can persist following primary infection, despite intervening antiretroviral therapy, and subsequently sexually transmitted. We provide "proof of principle" that such mutations can therefore become "fixed" within the circulating virus pool.